Methods and Compositions for Goss&#39; Wilt Resistance in Corn

ABSTRACT

The present invention relates to the field of plant breeding. More specifically, the present invention includes a method of using haploid plants for genetic mapping of traits of interest such as disease resistance. Further, the invention includes a method for breeding corn plants containing quantitative trait loci (QTL) that are associated with resistance to Goss&#39; Wilt, a bacterial disease associated with  Clavibacter michiganense  spp.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/294,351, filed Jun. 3, 2014, which is a Divison of U.S. patentapplication Ser. No. 13/742,042, filed Jan. 15, 2013, issued as U.S.Pat. No. 8,766,035, which is a continuation of U.S. patent applicationSer. No. 12/201,206, now abandoned, which claims the benefit of U.S.Provisional Patent Application No. 60/966,706, filed Aug. 29, 2007, eachof which are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing is contained in the file named“46_(—)25_(—)54886_(—)003_US_.txt” which is 2432172 bytes (measured inMS-Windows) and comprising 1,361 nucleotide sequences, created Jul. 14,2015, is electronically filed herewith and is incorporated herein byreference in its entirety.

FIELD OF INVENTION

The present invention relates to the field of plant breeding. Morespecifically, the present invention includes a method of using haploidplants for genetic mapping of traits such as disease resistance. Theinvention further includes a method for breeding corn plants containingQTL that are associated with Goss' Wilt, a bacterial disease associatedwith Clavibacter michiganense spp.

BACKGROUND OF INVENTION

Goss' Wilt, caused by the bacterial pathogen Clavibacter michiganensissubsp. nebraskensis (CN), is a disease that causes significant damage tocorn crops. Goss' Wilt has been identified throughout the U.S. CornBelt, primarily in the western regions. Symptoms include leaf freckleswhich are small dark green to black water soaked spots and vascular wiltwhich results in loss of yield. Conservation tillage practices canincrease pervasiveness because the bacterial pathogen Clavibactermichiganensis subsp. nebraskensis (CN) can overwinter in debris,particularly stalks, from infected corn plants (Bradbury, J. F. IMIdescription of Fungi and Bacteria, (1998)). A mapping study conducted byRocheford et al., reported a genomic region on maize Chromosome 4associated with Goss' Wilt (Rocheford, et al., Journal of Heredity80(5), (1989)). Goss' Wilt is a significant pathogen of corn, and a needexists for development of disease resistant lines.

Breeding for corn plants resistant to Goss' Wilt can be greatlyfacilitated by the use of marker-assisted selection. Of the classes ofgenetic markers, single nucleotide polymorphisms (SNPs) havecharacteristics which make them preferential to other genetic markers indetecting, selecting for, and introgressing disease resistance in a cornplant. SNPs are preferred because technologies are available forautomated, high-throughput screening of SNP markers, which can decreasethe time to select for and introgress disease resistance in corn plants.Further, SNP markers are ideal because the likelihood that a particularSNP allele is derived from independent origins in the extant populationof a particular species is very low. As such, SNP markers are useful fortracking and assisting introgression of disease resistance alleles,particularly in the case of disease resistance haplotypes.

The present invention further provides and includes a method forscreening and selecting a corn plant comprising QTL for Goss' Wiltresistance using endemic strains of CN and SNP marker technology.

SUMMARY OF THE INVENTION

Methods for identifying corn plants that comprise alleles of geneticloci associated with Goss' Wilt resistance are provided herein. Incertain embodiments, methods of identifying a corn plant comprising atleast one allele associated with Goss' Wilt resistance allele in a cornplant comprising: a) genotyping at least one corn plant with at leastone nucleic acid marker selected from the group consisting of SEQ IDNOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110,111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160,162, 164, 166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215,216, 218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260,265-267, 271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320,332-334, 337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392,395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447,474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556,566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630,632, 637, 639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690, 704,709, 710, 717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764,768, 773, 792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858,874, 876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100,1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146,1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229,1234-1302, and 1303, and b) selecting at least one corn plant comprisingan allele of at least one of the markers that is associated withresistance to Goss' Wilt are provided. In certain embodiments of themethods, the at least one corn plant genotyped in step (a) and/or the atleast one corn plant selected in step (b) is a corn plant from apopulation generated by a cross. In embodiments of the methods where thecorn plant from a population generated by a cross, the cross can beeffected by mechanical emasculation, chemical sterilization, or geneticsterilization of a pollen acceptor. In certain embodiments of themethods, genotyping is effected in step (a) by determining the allelicstate of at least one of the corn genomic DNA markers. In suchembodiments of the methods, an allelic state can be determined by singlebase extension (SBE), allele-specific primer extension sequencing(ASPE), DNA sequencing, RNA sequencing, microarray-based analyses,universal PCR, allele specific extension, hybridization, massspectrometry, ligation, extension-ligation, and/or a FlapEndonuclease-mediated assay(s). In other embodiments of the methods, theselected corn plant(s) of step (b) exhibit at least partial resistanceto a Goss' Wilt-inducing bacteria or at least substantial resistance toa Goss' Wilt-inducing bacteria. In certain embodiments of the methods,the nucleic acid marker is selected from the group consisting of SEQ IDNOs: 27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440, 479,480, 533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054,1122, 1186, 1246, 1250, and 1251. Alternatively, the nucleic acid markercan be selected from the group consisting of SEQ ID NOs: 234 and 1250.In embodiments where a population is generated by a cross, thepopulation can be generated by a cross of at least one Goss' Wiltresistant corn plant with at least one Goss' Wilt sensitive corn plant.In certain embodiments of the methods where a population is generated bya cross, the cross can be a back cross of at least one Goss' Wiltresistant corn plant with at least one Goss' Wilt sensitive corn plantto introgress Goss' Wilt resistance into a corn germplasm. Inembodiments where the corn plant is from a population, the populationcan be a segregating population. In certain embodiments of the methods,the population can be a haploid breeding population.

Also provided herein are corn plants obtained by any of theaforementioned methods of identifying corn plants that comprise allelesof genetic loci associated with Goss' Wilt resistance. In certainembodiments, a corn plant obtained by any of these aforementionedmethods can comprise at least one allele of a nucleic acid markerselected from the group consisting of SEQ ID NOs: 13, 19, 24, 27, 36,50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122, 124,128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166, 169, 172,175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220, 224, 228,231-236, 244, 248, 250, 252, 256, 257, 260, 265-267, 271-274, 278, 279,282, 287, 289, 294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363,366-368, 370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412,422, 423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589, 593,594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646, 649, 650,657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726,727, 733, 734, 744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821,825, 835, 844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893,896, 897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981,983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054,1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115,1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174,1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1302, and 1303, whereinthe allele is associated with Goss' Wilt resistance. In certainembodiments, a corn plant obtained by any of these aforementionedmethods can comprise at least one allele of a nucleic acid marker isselected from the group consisting of SEQ ID NOs: 27, 121, 141, 175,177, 220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582, 585, 639,721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122, 1186, 1246, 1250,and 1251, wherein the allele is associated with Goss' Wilt resistance.In certain embodiments, a corn plant obtained by any of theseaforementioned methods can comprise at least one allele of a nucleicacid marker is selected from the group consisting of SEQ ID NOs: 234 and1250, wherein the allele is associated with Goss' Wilt resistance. Incertain embodiments, a corn plant obtained by any of theseaforementioned methods exhibits at least partial resistance to a Goss'Wilt-inducing bacterium. In certain embodiments, a corn plant obtainedby any of these aforementioned methods exhibits at least substantialresistance to a Goss' Wilt-inducing bacterium. In still otherembodiments, a corn plant obtained by any of these aforementionedmethods can be a haploid corn plant. In certain embodiments, a cornplant obtained by any of the aforementioned methods can comprise atleast one transgenic trait. In such embodiments, the transgenic traitcan be herbicide tolerance and/or pest resistance. In embodiments wherethe corn plant obtained is herbicide tolerant, herbicide tolerance canbe selected from the group consisting of glyphosate, dicamba,glufosinate, sulfonylurea, bromoxynil and norflurazon herbicidetolerance. In certain embodiments, the nucleic acid marker is present asa single copy in a corn plant obtained by any of these aforementionedmethods. In other embodiments, the nucleic acid marker can be present intwo copies in a corn plant obtained by any of these aforementionedmethods.

Also provided are methods for introgressing a Goss' Wilt resistance QTLinto a corn plant. In certain embodiments, methods of introgressing aGoss' Wilt resistance QTL into a corn plant comprising: a) screening apopulation with at least one nucleic acid marker to determine if one ormore corn plants from the population contains a Goss' Wilt resistanceQTL, wherein the Goss' Wilt resistance QTL is a QTL selected from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, and 131 asprovided in FIG. 1; and b) selecting from the population at least onecorn plant comprising an allele of the marker associated with Goss' Wiltresistance are provided. In certain embodiments of the methods, at leastone of the markers is located within 30 cM, 25 cM, 20 cM, 15 cM, or 10cM of the Goss' Wilt resistance QTL. In other embodiments of themethods, at least one of the markers is located within 5 cM, 2 cM, or 1cM of the Goss' Wilt resistance QTL. In certain embodiments of themethods, at least one of the markers exhibits an LOD score of greaterthan 2.0, 2.5, or 3.0 with the Goss' Wilt resistance QTL. In otherembodiments of the methods, at least one of the markers exhibits a LODscore of greater than 4.0 with the Goss' Wilt resistance QTL. In certainembodiments of these methods, the nucleic acid marker is selected fromthe group consisting of SEQ ID NOs: 27, 121, 141, 175, 177, 220, 224,234, 248, 252, 381, 440, 479, 480, 533, 582, 585, 639, 721, 727, 733,746, 768, 773, 940, 1053, 1054, 1122, 1186, 1246, 1250, and 1251,wherein the nucleic acid marker is selected from the group consisting ofSEQ ID NOs: 234 and 1250. In certain embodiments of the methods, thepopulation is a segregating population.

Also provided herein are corn plants obtained by any of theaforementioned methods of identifying corn plants that comprise a Goss'Wilt resistance QTL. In certain embodiments, a corn plant obtained byany of the aforementioned methods is provided, wherein the corn plantcomprises a Goss' Wilt resistance QTL selected from the group consistingof QTL numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, and 131 asprovided in FIG. 1. In certain embodiments, a corn plant obtained by anyof these aforementioned methods and comprising at least one of the QTLexhibits at least partial resistance to a Goss' Wilt-inducing bacterium.In certain embodiments, a corn plant obtained by any of theseaforementioned methods exhibits at least substantial resistance to aGoss' Wilt-inducing bacterium. In still other embodiments, a corn plantobtained by any of these aforementioned methods and comprising at leastone of the QTL can be a haploid corn plant. In certain embodiments, acorn plant obtained by any of the aforementioned methods and comprisingat least one of the QTL can comprise at least one transgenic trait. Insuch embodiments, the transgenic trait can be herbicide tolerance and/orpest resistance. In embodiments where the corn plant obtained isherbicide tolerant, herbicide tolerance can be selected from the groupconsisting of glyphosate, dicamba, glufosinate, sulfonylurea, bromoxyniland norflurazon herbicide tolerance.

Also provided herein are isolated nucleic acid markers for identifyingpolymorphisms in corn DNA. These isolated nucleic acids can be used in avariety of applications, including but not limited to, theidentification of corn plants that comprise alleles of genetic lociassociated with Goss' Wilt resistance. In certain embodiments, anisolated nucleic acid molecule for detecting a molecular markerrepresenting a polymorphism in corn DNA, wherein the nucleic acidmolecule comprises at least 15 nucleotides that include or areimmediately adjacent to the polymorphism, wherein the nucleic acidmolecule is at least 90 percent identical to a sequence of the samenumber of consecutive nucleotides in either strand of DNA that includeor are immediately adjacent to the polymorphism, and wherein themolecular marker is selected from the group consisting of SEQ ID NOs:27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 440, 479, 480, 533,582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122,1186, 1234-1302, and 1303 is provided. In other embodiments, themolecular marker is selected from the group consisting of SEQ ID NOs:27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 381, 440, 479, 480,533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122,1186, 1246, 1250, and 1251. In still other embodiments, the molecularmarker is selected from the group consisting of SEQ ID NOs: 234 and1250. In certain embodiments, the isolated nucleic acid furthercomprises a detectable label or provides for incorporation of adetectable label. In such embodiments that comprise or provide forincorporation of a detectable label, the detectable label is selectedfrom the group consisting of an isotope, a fluorophore, an oxidant, areductant, a nucleotide and a hapten. In certain embodiments, thedetectable label is added to the nucleic acid by a chemical reaction oris incorporated by an enzymatic reaction. In certain embodiments, theisolated nucleic acid molecule comprises at least 16 or 17 nucleotidesthat include or are immediately adjacent to the polymorphism. In otherembodiments, the nucleic acid molecule comprises at least 18 nucleotidesthat include or are immediately adjacent to the polymorphism orcomprises at least 20 nucleotides that include or are immediatelyadjacent to the polymorphism. In certain embodiments, the isolatednucleic acid molecule hybridizes to at least one allele of the molecularmarker under stringent hybridization conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention.

In the drawings:

FIG. 1. Displays markers associated with resistance to Goss' Wilt. Thesymbol “*” represents a single nucleotide deletion.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The definitions and methods provided herein define the present inventionand guide those of ordinary skill in the art in the practice of thepresent invention. Unless otherwise noted, terms are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. Definitions of common terms in molecular biology may alsobe found in Alberts et al., Molecular Biolgoy of The Cell, 3rd Edition,Garland Publishing, Inc.: New York, 1994; Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

As used herein, a “locus” is a fixed position on a chromosome and mayrepresent a single nucleotide, a few nucleotides or a large number ofnucleotides in a genomic region.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to, one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismincludes a single nucleotide polymorphism (SNP), a simple sequencerepeat (SSR) and indels, which are insertions and deletions. Apolymorphism may arise from random processes in nucleic acidreplication, through mutagenesis, as a result of mobile genomicelements, from copy number variation and during the process of meiosis,such as unequal crossing over, genome duplication and chromosome breaksand fusions. The variation can be commonly found or may exist at lowfrequency within a population, the former having greater utility ingeneral plant breeding and the later may be associated with rare butimportant phenotypic variation.

As used herein, “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsmay include genetic markers, protein composition, protein levels, oilcomposition, oil levels, carbohydrate composition, carbohydrate levels,fatty acid composition, fatty acid levels, amino acid composition, aminoacid levels, biopolymers, pharmaceuticals, starch composition, starchlevels, fermentable starch, fermentation yield, fermentation efficiency,energy yield, secondary compounds, metabolites, morphologicalcharacteristics, and agronomic characteristics.

As used herein, “genetic marker” means polymorphic nucleic acid sequenceor nucleic acid feature. A “polymorphism” is a variation amongindividuals in sequence, particularly in DNA sequence, or feature, suchas a transcriptional profile or methylation pattern. Usefulpolymorphisms include single nucleotide polymorphisms (SNPs), insertionsor deletions in DNA sequence (Indels), simple sequence repeats of DNAsequence (SSRs) a restriction fragment length polymorphism, a haplotype,and a tag SNP. A genetic marker, a gene, a DNA-derived sequence, aRNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a3′ untranslated region of a gene, microRNA, siRNA, a QTL, a satellitemarker, a transgene, mRNA, ds mRNA, a transcriptional profile, and amethylation pattern may comprise polymorphisms.

As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.measurement of at least one phenotype (such as seed color, flower color,or other visually detectable trait), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies, andnucleic acid sequencing technologies, etc.

As used herein, the phrase “immediately adjacent”, when used to describea nucleic acid molecule that hybridizes to DNA containing apolymorphism, refers to a nucleic acid that hybridizes to DNA sequencesthat directly abut the polymorphic nucleotide base position. Forexample, a nucleic acid molecule that can be used in a single baseextension assay is “immediately adjacent” to the polymorphism.

As used herein, “interrogation position” refers to a physical positionon a solid support that can be queried to obtain genotyping data for oneor more predetermined genomic polymorphisms.

As used herein, “consensus sequence” refers to a constructed DNAsequence which identifies SNP and Indel polymorphisms in alleles at alocus. Consensus sequence can be based on either strand of DNA at thelocus and states the nucleotide base of either one of each SNP in thelocus and the nucleotide bases of all Indels in the locus. Thus,although a consensus sequence may not be a copy of an actual DNAsequence, a consensus sequence is useful for precisely designing primersand probes for actual polymorphisms in the locus.

As used herein, the term “single nucleotide polymorphism,” also referredto by the abbreviation “SNP,” means a polymorphism at a single sitewherein said polymorphism constitutes a single base pair change, aninsertion of one or more base pairs, or a deletion of one or more basepairs.

As used herein, “genotype” means the genetic component of the phenotypeand it can be indirectly characterized using markers or directlycharacterized by nucleic acid sequencing. Suitable markers include aphenotypic character, a metabolic profile, a genetic marker, or someother type of marker. A genotype may constitute an allele for at leastone genetic marker locus or a haplotype for at least one haplotypewindow. In some embodiments, a genotype may represent a single locus andin others it may represent a genome-wide set of loci. In anotherembodiment, the genotype can reflect the sequence of a portion of achromosome, an entire chromosome, a portion of the genome, and theentire genome.

As used herein, the term “haplotype” means a chromosomal region within ahaplotype window defined by at least one polymorphic molecular marker.The unique marker fingerprint combinations in each haplotype windowdefine individual haplotypes for that window. Further, changes in ahaplotype, brought about by recombination for example, may result in themodification of a haplotype so that it comprises only a portion of theoriginal (parental) haplotype operably linked to the trait, for example,via physical linkage to a gene, QTL, or transgene. Any such change in ahaplotype would be included in our definition of what constitutes ahaplotype so long as the functional integrity of that genomic region isunchanged or improved.

As used herein, the term “haplotype window” means a chromosomal regionthat is established by statistical analyses known to those of skill inthe art and is in linkage disequilibrium. Thus, identity by statebetween two inbred individuals (or two gametes) at one or more molecularmarker loci located within this region is taken as evidence ofidentity-by-descent of the entire region. Each haplotype window includesat least one polymorphic molecular marker. Haplotype windows can bemapped along each chromosome in the genome. Haplotype windows are notfixed per se and, given the ever-increasing density of molecularmarkers, this invention anticipates the number and size of haplotypewindows to evolve, with the number of windows increasing and theirrespective sizes decreasing, thus resulting in an ever-increasing degreeconfidence in ascertaining identity by descent based on the identity bystate at the marker loci.

As used herein, a plant referred to as “haploid” has a single set(genome) of chromosomes and the reduced number of chromosomes (n) in thehaploid plant is equal to that of the gamete.

As used herein, a plant referred to as “doubled haploid” is developed bydoubling the haploid set of chromosomes. A plant or seed that isobtained from a doubled haploid plant that is selfed any number ofgenerations may still be identified as a doubled haploid plant. Adoubled haploid plant is considered a homozygous plant. A plant isconsidered to be doubled haploid if it is fertile, even is the entirevegetative part of the plant does not consist of the cells with thedoubled set of chromosomes; that is, a plant will be considered doubledhaploid if it contains viable gametes, even if it is chimeric.

As used herein, a plant referred to as “diploid” has two sets (genomes)of chromosomes and the chromosome number (2n) is equal to that of thezygote.

As used herein, the term “plant” includes whole plants, plant organs(i.e., leaves, stems, roots, etc.), seeds, and plant cells and progenyof the same. “Plant cell” includes without limitation seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, shoots,gametophytes, sporophytes, pollen, and microspores.

As used herein, a “genetic map” is the ordered list of loci known for aparticular genome.

As used herein, “phenotype” means the detectable characteristics of acell or organism which are a manifestation of gene expression.

As used herein, a “phenotypic marker” refers to a marker that can beused to discriminate phenotypes displayed by organisms.

As used herein, “linkage” refers to relative frequency at which types ofgametes are produced in a cross. For example, if locus A has genes “A”or “a” and locus B has genes “B” or “b” and a cross between parent Iwith AABB and parent B with aabb will produce four possible gameteswhere the genes are segregated into AB, Ab, aB and ab. The nullexpectation is that there will be independent equal segregation intoeach of the four possible genotypes, i.e. with no linkage ¼ of thegametes will of each genotype. Segregation of gametes into a genotypesdiffering from ¼ are attributed to linkage.

As used herein, “linkage disequilibrium” is defined in the context ofthe relative frequency of gamete types in a population of manyindividuals in a single generation. If the frequency of allele A is p, ais p′, B is q and b is q′, then the expected frequency (with no linkagedisequilibrium) of genotype AB is pq, Ab is pq′, aB is p′q and ab isp′q′. Any deviation from the expected frequency is called linkagedisequilibrium. Two loci are said to be “genetically linked” when theyare in linkage disequilibrium.

As used herein, “quantitative trait locus (QTL)” means a locus thatcontrols to some degree numerically representable traits that areusually continuously distributed.

As used herein, the term “transgene” means nucleic acid molecules inform of DNA, such as cDNA or genomic DNA, and RNA, such as mRNA ormicroRNA, which may be single or double stranded.

As used herein, the term “inbred” means a line that has been bred forgenetic homogeneity.

As used herein, the term “hybrid” means a progeny of mating between atleast two genetically dissimilar parents. Without limitation, examplesof mating schemes include single crosses, modified single cross, doublemodified single cross, three-way cross, modified three-way cross, anddouble cross wherein at least one parent in a modified cross is theprogeny of a cross between sister lines.

As used herein, the term “tester” means a line used in a testcross withanother line wherein the tester and the lines tested are from differentgermplasm pools. A tester may be isogenic or nonisogenic.

As used herein, “resistance allele” means the isolated nucleic acidsequence that includes the polymorphic allele associated with resistanceto the disease or condition of concern.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, an “elite line” is any line that has resulted frombreeding and selection for superior agronomic performance.

As used herein, an “inducer” is a line which when crossed with anotherline promotes the formation of haploid embryos.

As used herein, “haplotype effect estimate” means a predicted effectestimate for a haplotype reflecting association with one or morephenotypic traits, wherein the associations can be made de novo or byleveraging historical haplotype-trait association data.

As used herein, “breeding value” means a calculation based on nucleicacid sequence effect estimates and nucleic acid sequence frequencyvalues, the breeding value of a specific nucleic acid sequence relativeto other nucleic acid sequences at the same locus (i.e., haplotypewindow), or across loci (i.e., haplotype windows), can also bedetermined. In other words, the change in population mean by fixing saidnucleic acid sequence is determined. In addition, in the context ofevaluating the effect of substituting a specific region in the genome,either by introgression or a transgenic event, breeding values providethe basis for comparing specific nucleic acid sequences for substitutioneffects. Also, in hybrid crops, the breeding value of nucleic acidsequences can be calculated in the context of the nucleic acid sequencein the tester used to produce the hybrid.

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein or in any reference found elsewhere, it isunderstood that the preceding definition will be used herein.

Methods and Compositions for Goss' Wilt Resistance in Corn

The present invention provides a method of using haploid plants toidentify genotypes associated with phenotypes of interest wherein thehaploid plant is assayed with at least one marker and associating the atleast one marker with at least one phenotypic trait. The genotype ofinterest can then be used to make decisions in a plant breeding program.Such decisions include, but are not limited to, selecting among newbreeding populations which population has the highest frequency offavorable nucleic acid sequences based on historical genotype andagronomic trait associations, selecting favorable nucleic acid sequencesamong progeny in breeding populations, selecting among parental linesbased on prediction of progeny performance, and advancing lines ingermplasm improvement activities based on presence of favorable nucleicacid sequences. Non-limiting examples of germplasm improvementactivities include line development, hybrid development, transgenicevent selection, making breeding crosses, testing and advancing a plantthrough self fertilization, using plants for transformation, usingplants for candidates for expression constructs, and using plants formutagenesis.

Non-limiting examples of breeding decisions include progeny selection,parent selection, and recurrent selection for at least one haplotype. Inanother aspect, breeding decisions relating to development of plants forcommercial release comprise advancing plants for testing, advancingplants for purity, purification of sublines during development, inbreddevelopment, variety development, and hybrid development. In yet otheraspects, breeding decisions and germplasm improvement activitiescomprise transgenic event selection, making breeding crosses, testingand advancing a plant through self-fertilization, using plants fortransformation, using plants for candidates for expression constructs,and using plants for mutagenesis.

In still another embodiment, the present invention acknowledges thatpreferred haplotypes and QTL identified by the methods presented hereinmay be advanced as candidate genes for inclusion in expressionconstructs, i.e., transgenes. Nucleic acids underlying haplotypes or QTLof interest may be expressed in plant cells by operably linking them toa promoter functional in plants. In another aspect, nucleic acidsunderlying haplotypes or QTL of interest may have their expressionmodified by double-stranded RNA-mediated gene suppression, also known asRNA interference (“RNAi”), which includes suppression mediated by smallinterfering RNAs (“siRNA”), trans-acting small interfering RNAs(“ta-siRNA”), or microRNAs (“miRNA”). Examples of RNAi methodologysuitable for use in plants are described in detail in U.S. patentapplication publications 2006/0200878 and 2007/0011775.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the nucleic acid molecule for a traitis transcribed into a functional mRNA molecule that is translated andexpressed as a protein product. For the practice of the presentinvention, conventional compositions and methods for preparing and usingconstructs and host cells are well known to one skilled in the art, seefor example, Molecular Cloning: A Laboratory Manual, 3rd Edition Volumes1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, ColdSpring Harbor Laboratory Press. Methods for making transformationconstructs particularly suited to plant transformation include, withoutlimitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835,4,769,061 and 4,757,011, all of which are herein incorporated byreference in their entirety. Transformation methods for the introductionof expression units into plants are known in the art and includeelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; protoplasttransformation as illustrated in U.S. Pat. No. 5,508,184; andAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301.

The present invention provides Goss' Wilt resistance loci that arelocated in public bins in the maize genome that were not previouslyassociated with Goss' Wilt resistance.

The present invention provides 130 Goss' Wilt resistance loci that arelocated in public bins in the maize genome that were not previouslyassociated with Goss' Wilt resistance. QTL were assigned by dividingmaize chromosomal regions into 10 cM windows. A total of 131 QTL wereidentified, with 130 not having been previously reported. SNP markersare also provided for monitoring the introgression of the 131 QTLassociated with Goss' Wilt resistance.

In the present invention, Goss' Wilt resistance loci 1-53 and 55-131have not been previously associated with Goss' Wilt and are provided.SNP markers are also provided for monitoring the introgression of Goss'Wilt resistance. In the present invention, Goss' Wilt resistance loci 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20are located on Chromosome 1. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 1 include those selectedfrom the group consisting of SEQ ID NOs: 13 and 1274. SNP markers usedto monitor the introgression of Goss' Wilt resistance locus 2 includedthose selected from the group consisting of SEQ ID NOs: 1234 and 19. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus3 include those selected from the group consisting of SEQ ID NOs: 27 and24. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 4 include SEQ ID NO: 36. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 5 included thoseselected from the group consisting of SEQ ID NOs: 50 and 53. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 6include SEQ ID NO: 90. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 7 include those selected from the groupconsisting of SEQ ID NOs: 94, 95, 97, 1235, 1236, 99, 101, 102, 1237,106, 1238, 110, 111, and 1239. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 8 include those selectedfrom the group consisting of SEQ ID NOs: 1240, 119, 121, 122, and 124.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 9 include those selected from the group consisting of SEQ ID NOs:128, 130, 131, and 132. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 10 include those selected from the groupconsisting of SEQ ID NOs: 136, 138, and 1275. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 11 include SEQID NOs: 141. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 12 include SEQ ID NOs: 146. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 13 include thoseselected from the group consisting of SEQ ID NOs: 153, 1241, 159, 160,162, and 158. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 14 include those selected from the groupconsisting of SEQ ID NOs: 164, 166, 169, and 172. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 15 includethose selected from the group consisting of SEQ ID NOs: 175 and 177. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus16 include those selected from the group consisting of SEQ ID NOs: 1242and 186. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 17 include SEQ ID NO: 200. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 18 include thoseselected from the group consisting of SEQ ID NOs: 202 and 203. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus19 include those selected from the group consisting of SEQ ID NOs: 207and 208. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 20 include SEQ ID NO: 1243.

In the present invention Goss' Wilt resistance loci 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 129 are located onChromosome 2. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 21 include SEQ ID NO: 215. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 22 includethose selected from the group consisting of SEQ ID NOs: 216, 1244, 220,218, and 1229. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 23 include those selected from the groupconsisting of SEQ ID NOs: 224. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 24 include those selectedfrom the group consisting of SEQ ID NO: 228, 231, and 1276. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 25include those selected from the group consisting of SEQ ID NOs: 232,233, 234, 235, and 236. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 26 include those selected from the groupconsisting of SEQ ID NOs: 244, 248, and 1277. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 27 includethose selected from the group consisting of SEQ ID NOs: 250, 252, 256,257, 260, 1295, and 1278. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 28 include those selected from the groupconsisting of SEQ ID NOs: 265, 266, and 267. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 29 include thoseselected from the group consisting of SEQ ID NOs: 271, 273, 1245, 274,278, 279, 282, 287, 289, and 272. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 30 include those selectedfrom the group consisting of SEQ ID NOs: 294, 295, 296, and 299. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus31 include those selected from the group consisting of SEQ ID NOs: 1246,317, and 320. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 32 include those selected from the groupconsisting of SEQ ID NOs: 332, 333, and 334. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 33 include SEQ ID NO:337. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 34 include SEQ ID NO: 347. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 35 include SEQ ID NO:355. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 129 include SEQ ID NO: 1294.

In the present invention Goss' Wilt resistance loci 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 122, and 123 are located onChromosome 3. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 36 include those selected from the groupconsisting of SEQ ID NOs: 362 and 363. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 37 include SEQ ID NO: 1247.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 38 include SEQ ID NO: 366. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 39 include those selectedfrom the group consisting of SEQ ID NO: 367, 368, and 1279. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 40include those selected from the group consisting of SEQ ID NO: 370 and371. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 41 include those selected from the group consisting ofSEQ ID NOs: 381, 382, 392, and 395. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 42 include those selectedfrom the group consisting of SEQ ID NOs: 409, 411, and 412. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 43include those selected from the group consisting of SEQ ID NOs: 419,422, 423, and 1280. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 44 include those selected from the groupconsisting of SEQ ID NOs: 429, 430, 433 and 1281. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 45 includethose selected from the group consisting of SEQ ID NOs: 438, 440, 1248,and 447. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 46 include SEQ ID NO: 1249. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 47 include thoseselected from the group consisting of SEQ ID NOs: 474, 476, 479, 480,and 482. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 48 include those selected from the group SEQ ID NO:486. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 49 include SEQ ID NO: 490. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 50 include SEQ ID NO:493. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 122 include those selected from the group consisting ofSEQ ID NOs: 375 and 1296. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 123 include those selected from the groupconsisting of SEQ ID NOs: 401 and 408.

In the present invention Goss' Wilt resistance loci 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 124, 125, and 126 are located on Chromosome 4.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 51 include SEQ ID NO: 500. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 52 include those selectedfrom the group consisting of SEQ ID NOs: 1250, 525, and 530. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 53include SEQ ID NOs: 533. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 54 include those selected from the groupconsisting of SEQ ID NOs: 556 and 566. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 55 include those selectedfrom the group consisting of SEQ ID NOs: 582, 585, 1251, and 1283. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus56 include those selected from the group consisting of SEQ ID NOs: 589and 587. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 57 include those selected from the group consisting ofSEQ ID NOs: 593, 594, and 599. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 58 include SEQ ID NO: 611,1297, 1298, and 1284. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 59 include those selected from the groupconsisting of SEQ ID NOs: 1252, 618, 621, and 623. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 60 includethose selected from the group consisting of SEQ ID NOs: 630, 632, 637,639, and 629. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 61 include those selected from the groupconsisting of SEQ ID NOs: 646, 649, and 650. SNP markers used to monitorthe introgression of Goss' Wilt resistance locus 124 include SEQ ID NO:498. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 125 include SEQ ID NO: 1282.

In the present invention Goss' Wilt resistance loci 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, and 130 are located on Chromosome 5.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 62 include SEQ ID NO: 657. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 63 include those selectedfrom the group consisting of SEQ ID NOs: 665, 1286, and 1299. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus64 include SEQ ID NO: 669. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 65 include those selected from SEQ ID NO:1253. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 66 include those selected from the group consisting ofSEQ ID NOs: 678, 1254, and 1255. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 67 include those selectedfrom the group consisting of SEQ ID NOs: 679, 688, and 690. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 68include those selected from the group consisting of SEQ ID NOs: 1256,704, 709, and 1300. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 69 include those selected from the groupconsisting of SEQ ID NOs: 710, 717, 719, 720, 1257, and 721. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 70include those selected from the group SEQ ID NOs: 726, 727, and 1258.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 71 include those selected from the group consisting of SEQ ID NOs:733 and 734. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 72 include those selected from the group consisting ofSEQ ID NOs: 746 and 744. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 73 include those selected from the groupconsisting of SEQ ID NOs: 758 and 760. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 74 include those selectedfrom the group consisting of SEQ ID NOs: 764, 768, and 1287. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 75include SEQ ID NO: 773. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 130 include SEQ ID NO: 1301.

In the present invention Goss' Wilt resistance loci 76, 77, 78, 79, 80,81, 82, 83, and 84 are located on Chromosome 6. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 76 includethose selected from the group consisting of SEQ ID NOs: 1259, 792, 793,and 812. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 77 include those selected from the group consisting ofSEQ ID NOs: 821 and 825. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 78 include those selected from the groupconsisting of SEQ ID NOs: 835, 1260, and 844. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 79 includethose selected from the group consisting of SEQ ID NOs: 846, 850, and854. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 80 include those selected from the group consisting ofSEQ ID NOs: 856, 857, and 858. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 81 include SEQ ID NO: 1261.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 82 include SEQ ID NO: 874. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 83 include those selectedfrom the group consisting of SEQ ID NOs: 876, 880, and 882. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 84include SEQ ID NO: 885.

In the present invention Goss' Wilt resistance loci 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, and 127 are located on Chromosome 7. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus85 include SEQ ID NO: 893. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 86 include those selected from the groupconsisting of SEQ ID NOs: 897 and 896. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 87 include SEQ ID NO: 1262.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 88 include those selected from the group consisting of SEQ ID NOs:915, 926, and 1288. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 89 include those selected from the groupconsisting of SEQ ID NOs: 940 and 942. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 90 include those selectedfrom the group consisting of SEQ ID NOs: 949 and 951. SNP markers usedto monitor the introgression of Goss' Wilt resistance locus 91 includeSEQ ID NO: 957 and 963. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 92 include those selected from the groupconsisting of SEQ ID NO: 964 and 1289. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 93 include those selectedfrom the group consisting of SEQ ID NO: 974 and 976. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 94 include SEQID NO: 1263. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 95 include those selected from the group consisting ofSEQ ID NO: 981 and 1291. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 96 include SEQ ID NOs: 983 and 990. SNPmarkers used to monitor the introgression of Goss' Wilt resistance locus127 include SEQ ID NO: 1290.

In the present invention Goss' Wilt resistance loci 97, 98, 99, 100,101, 102, 103, and 131 are located on Chromosome 8. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 97 includethose selected from the group consisting of SEQ ID NOs: 997, 999, and1000. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 98 include those selected from the group consisting ofSEQ ID NOs: 1016 and 1264. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 99 include those selected from the groupconsisting of SEQ ID NOs: 1027, 1265, and 1303. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 100 include SEQID NO: 1043. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 101 include SEQ ID NO: 1049. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 102 include SEQID NO: 1056. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 103 include SEQ ID NO: 1075. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 131 include SEQID NO: 1015.

In the present invention Goss' Wilt resistance loci 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, and 115 are located on Chromosome 9.SNP markers used to monitor the introgression of Goss' Wilt resistancelocus 104 include those selected from the group consisting of SEQ IDNOs: 1266 and 1081. SNP markers used to monitor the introgression ofGoss' Wilt resistance locus 105 include SEQ ID NO: 1087. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 106include SEQ ID NO: 1088. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 107 include SEQ ID NO: 1098. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 108include those selected from the group consisting of SEQ ID NOs: 1099,1100, 1104, 1105, 1108, 1110, and 1292. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 109 include those selectedfrom the group consisting of SEQ ID NOs: 1267 and 1115. SNP markers usedto monitor the introgression of Goss' Wilt resistance locus 110 includethose selected from the group consisting of SEQ ID NOs: 1122, 1268,1131, and 1133. SNP markers used to monitor the introgression of Goss'Wilt resistance locus 111 include those selected from the groupconsisting of SEQ ID NOs: 1269 and 1142. SNP markers used to monitor theintrogression of Goss' Wilt resistance locus 112 include those selectedfrom the group consisting of SEQ ID NOs: 1143, 1145, 1146, 1148, and1149. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 113 include SEQ ID NO: 1270. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 114 include SEQID NO: 1159.

In the present invention Goss' Wilt resistance loci 115, 116, 117, 118,119, 120, 121, and 122 are located on Chromosome 10. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 115 include SEQID NO: 1168. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 116 include SEQ ID NO: 1174. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 117 includethose selected from the group consisting of SEQ ID NOs: 1271, 1184, and1186. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 118 include those selected from the group consisting ofSEQ ID NO: 1272 and 1196. SNP markers used to monitor the introgressionof Goss' Wilt resistance locus 119 include SEQ ID NO: 1204. SNP markersused to monitor the introgression of Goss' Wilt resistance locus 120include those selected from the group consisting of SEQ ID NOs: 1212 and1215. SNP markers used to monitor the introgression of Goss' Wiltresistance locus 121 include SEQ ID NO: 1273. SNP markers used tomonitor the introgression of Goss' Wilt resistance locus 128 include SEQID NO: 1293.

Exemplary marker assays for screening for Goss' Wilt resistance loci areprovided in Tables 3, 4, and 5. Illustrative Goss' Wilt resistance locus87 SNP marker DNA sequence SEQ ID NO: 896 can be amplified using theprimers indicated as SEQ ID NOs: 1332 through 1333 and detected withprobes indicated as SEQ ID NOs: 1334 through 1335. Illustrative Goss'Wilt resistance locus 91 SNP marker DNA sequence SEQ ID NO: 951 can beamplified using the primers indicated as SEQ ID NOs: 1336 through 1337and detected with probes indicated as SEQ ID NOs: 1338 through 1339.Illustrative Goss' Wilt resistance locus 72 SNP marker DNA sequence SEQID NO: 733 can be amplified using the primers indicated as SEQ ID NOs:1340 through 1341 and detected with probes indicated as SEQ ID NOs: 1342through 1343. Illustrative Goss' Wilt resistance locus 109 SNP markerDNA sequence SEQ ID NO: 1098 can be amplified using the primersindicated as SEQ ID NOs: 1344 through 1345 and detected with probesindicated as SEQ ID NOs: 1346 through 1347. Illustrative oligonucleotidehybridization probes for Goss' Wilt resistance locus 87 SNP marker DNAsequence SEQ ID NO: 896 are provided as SEQ ID NO: 1348 and SEQ ID NO1349. Illustrative oligonucleotide hybridization probes for Goss' Wiltresistance locus 91 SNP marker DNA sequence SEQ ID NO: 951 are providedas SEQ ID NO: 1350 and SEQ ID NO 1351. Illustrative oligonucleotidehybridization probes for Goss' Wilt resistance locus 72 SNP marker DNAsequence SEQ ID NO: 733 are provided as SEQ ID NO: 1352 and SEQ ID NO1353. Illustrative oligonucleotide hybridization probes for Goss' Wiltresistance locus 109 SNP marker DNA sequence SEQ ID NO: 1098 areprovided as SEQ ID NO: 1354 and SEQ ID NO 1355. An illustrative probefor single base extension assays for Goss' Wilt resistance locus 87 SNPmarker DNA sequence SEQ ID NO: 896 is provided as SEQ ID NO: 1356. Anillustrative probe for single base extension assays for Goss' Wiltresistance locus 91 SNP marker DNA sequence SEQ ID NO: 951 is providedas SEQ ID NO: 1357. An illustrative probe for single base extensionassays for Goss' Wilt resistance locus 72 SNP marker DNA sequence SEQ IDNO: 733 is provided as SEQ ID NO: 1358. An illustrative probe for singlebase extension assays for Goss' Wilt resistance locus 109 SNP marker DNAsequence SEQ ID NO: 1098 is provided as SEQ ID NO: 1359.

As used herein, Goss' Wilt refers to any Goss' Wilt variant or isolate.A corn plant of the present invention can be resistant to one or morebacteria capable of causing or inducing Goss' Wilt. In one aspect, thepresent invention provides plants resistant to Goss' Wilt as well asmethods and compositions for screening corn plants for resistance orsusceptibility to Goss' Wilt, caused by the genus Clavibacter. In apreferred aspect, the present invention provides methods andcompositions for screening corn plants for resistance or susceptibilityto Clavibacter michiganense spp.

In an aspect, the plant is selected from the genus Zea. In anotheraspect, the plant is selected from the species Zea mays. In a furtheraspect, the plant is selected from the subspecies Zea mays L. ssp. mays.In an additional aspect, the plant is selected from the group Zea maysL. subsp. mays Indentata, otherwise known as dent corn. In anotheraspect, the plant is selected from the group Zea mays L. subsp. maysIndurata, otherwise known as flint corn. In an aspect, the plant isselected from the group Zea mays L. subsp. mays Saccharata, otherwiseknown as sweet corn. In another aspect, the plant is selected from thegroup Zea mays L. subsp. mays Amylacea, otherwise known as flour corn.In a further aspect, the plant is selected from the group Zea mays L.subsp. mays Everta, otherwise known as pop corn. Zea plants includehybrids, inbreds, partial inbreds, or members of defined or undefinedpopulations.

Plants of the present invention can be a corn plant that is veryresistant, resistant, substantially resistant, mid-resistant,comparatively resistant, partially resistant, mid-susceptible, orsusceptible.

In a preferred aspect, the present invention provides a corn plant to beassayed for resistance or susceptibility to Goss' Wilt by any method todetermine whether a corn plant is very resistant, resistant,substantially resistant, mid-resistant, comparatively resistant,partially resistant, mid-susceptible, or susceptible.

Phenotyping for Goss' Wilt is based on visually screening plants todetermine percentage of infected leaf area. The percentage of leaf areainfected is used to rate plants on a scale of 1 (very resistant) to 9(susceptible).

A disease resistance QTL of the present invention may be introduced intoan elite corn inbred line.

In another aspect, the corn plant can show a comparative resistancecompared to a non-resistant control corn plant. In this aspect, acontrol corn plant will preferably be genetically similar except for theGoss' Wilt resistant allele or alleles in question. Such plants can begrown under similar conditions with equivalent or near equivalentexposure to the pathogen. In this aspect, the resistant plant or plantshas less than 25%, 15%, 10%, 5%, 2% or 1% of leaf area infected.

A disease resistance QTL of the present invention may be introduced intoan elite corn inbred line. An “elite line” is any line that has resultedfrom breeding and selection for superior agronomic performance.

A Goss' Wilt resistance QTL of the present invention may also beintroduced into an elite corn plant comprising one or more transgenesconferring herbicide tolerance, increased yield, insect control, fungaldisease resistance, virus resistance, nematode resistance, bacterialdisease resistance, mycoplasma disease resistance, modified oilsproduction, high oil production, high protein production, germinationand seedling growth control, enhanced animal and human nutrition, lowraffinose, environmental stress resistant, increased digestibility,industrial enzymes, pharmaceutical proteins, peptides and smallmolecules, improved processing traits, improved flavor, nitrogenfixation, hybrid seed production, reduced allergenicity, biopolymers,and biofuels among others. In one aspect, the herbicide tolerance isselected from the group consisting of glyphosate, dicamba, glufosinate,sulfonylurea, bromoxynil and norflurazon herbicides. These traits can beprovided by methods of plant biotechnology as transgenes in corn.

A disease resistant QTL allele or alleles can be introduced from anyplant that contains that allele (donor) to any recipient corn plant. Inone aspect, the recipient corn plant can contain additional Goss' Wiltresistant loci. In another aspect, the recipient corn plant can containa transgene. In another aspect, while maintaining the introduced QTL,the genetic contribution of the plant providing the disease resistantQTL can be reduced by back-crossing or other suitable approaches. In oneaspect, the nuclear genetic material derived from the donor material inthe corn plant can be less than or about 50%, less than or about 25%,less than or about 13%, less than or about 5%, 3%, 2% or 1%, but thatgenetic material contains the Goss' Wilt resistant locus or loci ofinterest.

It is further understood that a corn plant of the present invention mayexhibit the characteristics of any relative maturity group. In anaspect, the maturity group is selected from the group consisting ofRM90-95, RM 95-100, RM 100-105, RM 105-110, RM 110-115, and RM 115-120.

The present invention also includes a method of introgressing an alleleinto a corn plant comprising: (A) crossing at least one Goss' Wiltresistant corn plant with at least one Goss' Wilt sensitive corn plantin order to form a segregating population; (B) screening the segregatingpopulation with one or more nucleic acid markers to determine if one ormore corn plants from the segregating population contains a Goss' Wiltresistant allele, wherein the Goss' Wilt resistant allele is an alleleselected from the group consisting of Goss' Wilt resistant locus 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 132,124, 125, 126, 127, 128, 129, 130, and Goss' Wilt resistant locus 131.

The present invention includes isolated nucleic acid molecules. Suchmolecules include those nucleic acid molecules capable of detecting apolymorphism genetically or physically linked to a Goss' Wilt locus.Such molecules can be referred to as markers. Additional markers can beobtained that are linked to Goss' Wilt resistance locus 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, and Goss' Wilt resistant locus 131 by availabletechniques. In one aspect, the nucleic acid molecule is capable ofdetecting the presence or absence of a marker located less than 30, 20,10, 5, 2, or 1 centimorgans from a Goss' Wilt resistance locus. Inanother aspect, a marker exhibits a LOD score of 2 or greater, 3 orgreater, or 4 or greater with Goss' Wilt, measuring using Qgene Version2.23 (1996) and default parameters. In another aspect, the nucleic acidmolecule is capable of detecting a marker in a locus selected from thegroup Goss' Wilt resistance locus 1 through resistance locus 131. In afurther aspect, a nucleic acid molecule is selected from the groupconsisting of SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97,99, 101, 102, 106, 110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138,141, 146, 153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200,202, 203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368, 370,371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422, 423, 429,430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486, 490, 493, 498,500, 525, 530, 533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611,618, 621, 623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669,678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835, 844,846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915,926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981, 983, 990, 997,999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081,1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133,1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,1204, 1212, 1215, 1229, 1234-1303, 1332-1359 fragments thereof,complements thereof, and nucleic acid molecules capable of specificallyhybridizing to one or more of these nucleic acid molecules.

In a preferred aspect, a nucleic acid molecule of the present inventionincludes those that will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NOs: 13, 19, 24, 27, 36, 50,53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121, 122, 124,128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166, 169, 172,175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220, 224, 228,231-236, 244, 248, 250, 252, 256, 257, 260, 265-267, 271-274, 278, 279,282, 287, 289, 294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363,366-368, 370, 371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412,422, 423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486,490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587, 589, 593,594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646, 649, 650,657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726,727, 733, 734, 744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821,825, 835, 844, 846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893,896, 897, 915, 926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981,983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054,1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115,1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174,1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303, 1332-1359 orcomplements thereof or fragments of either under moderately stringentconditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred aspect, a nucleic acid of the present inventionwill specifically hybridize to one or more of the nucleic acid moleculesset forth in SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99,101, 102, 106, 110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138,141, 146, 153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200,202, 203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368, 370,371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422, 423, 429,430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486, 490, 493, 498,500, 525, 530, 533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611,618, 621, 623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669,678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835, 844,846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915,926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981, 983, 990, 997,999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081,1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133,1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,1204, 1212, 1215, 1229, 1234-1303, 1332-1359 or complements or fragmentsof either under high stringency conditions. In one aspect of the presentinvention, a preferred marker nucleic acid molecule of the presentinvention has the nucleic acid sequence set forth in SEQ ID NOs: 13, 19,24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119,121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164,166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218,220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337,347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401,408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474, 476,479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556, 566, 582,585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637,639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710,717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874, 876,880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951, 957, 963,964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043,1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105,1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149,1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,1332-1359 or complements thereof or fragments of either. In anotheraspect of the present invention, a preferred marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with the nucleic acid sequences set forth in SEQID NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106,110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153,158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200, 202, 203, 207,208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256,257, 260, 265-267, 271-274, 278, 279, 282, 287, 289, 294-296, 299, 317,320, 332-334, 337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382,392, 395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440,447, 474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533,556, 566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629,630, 632, 637, 639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690,704, 709, 710, 717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760,764, 768, 773, 792, 793, 812, 821, 825, 835, 844, 846, 850, 854,856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942,949, 951, 957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015,1016, 1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088,1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143,1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212,1215, 1229, 1234-1303, 1332-1359 or complements thereof or fragments ofeither. In a further aspect of the present invention, a preferred markernucleic acid molecule of the present invention shares between 95% and100% sequence identity with the sequences set forth in SEQ ID NOs: 13,19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111,119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162,164, 166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216,218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337,347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401,408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474, 476,479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556, 566, 582,585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637,639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710,717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874, 876,880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951, 957, 963,964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043,1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105,1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149,1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,1332-1359 or complements thereof or fragments of either. In a morepreferred aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 98% and 100%sequence identity with the nucleic acid sequence set forth in SEQ IDNOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110,111, 119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160,162, 164, 166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215,216, 218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260,265-267, 271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320,332-334, 337, 347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392,395, 401, 408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447,474, 476, 479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556,566, 582, 585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630,632, 637, 639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690, 704,709, 710, 717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764,768, 773, 792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858,874, 876, 880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951,957, 963, 964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016,1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100,1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146,1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229,1234-1303, 1332-1359 or complement thereof or fragments of either.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules are exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12:203-213; andWetmur et al., 1968 J. Mol. Biol. 31:349-370. Appropriate stringencyconditions that promote DNA hybridization are, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,1989, 6.3.1-6.3.6. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

For example, hybridization using DNA or RNA probes or primers can beperformed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.

A fragment of a nucleic acid molecule provided herein can be of anysize. Fragments provided herein include, but are not limited to,fragments of nucleic acid sequences set forth in SEQ ID NOs: 13, 19, 24,27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111, 119, 121,122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162, 164, 166,169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216, 218, 220,224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267, 271-274,278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337, 347, 355,362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401, 408, 409,411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474, 476, 479, 480,482, 486, 490, 493, 498, 500, 525, 530, 533, 556, 566, 582, 585, 587,589, 593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637, 639, 646,649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710, 717,719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773, 792,793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874, 876, 880,882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951, 957, 963, 964,974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043, 1049,1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105, 1108,1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149, 1159,1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1303,1332-1359 and complements thereof. In one aspect, a fragment of anucleic acid molecule can be 15 to 25, 15 to 30, 15 to 40, 15 to 50, 15to 100, 20 to 25, 20 to 30, 20 to 40, 20 to 50, 20 to 100, 25 to 30, 25to 40, 25 to 50, 25 to 100, 30 to 40, 30 to 50, or 30 to 100 nucleotidesin length. In another aspect, the fragment can be greater than 10, 15,20, 25, 30, 35, 40, 50, 100, or 250 nucleotides in length.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with Goss' Wilt resistance. Examples of publicmarker databases include, but are not limited to, the Maize GenomeDatabase located on the world wide web at www.maizegdb.org, the MaizeSeqdatabase located on the world wide web at www.www.maizeseq.org, thePanzea maize marker and map database located on the world wide web atwww.panzea.org, and the MAGI database located on the world wide web atwww.plantgenomics.iastate.edu/maize.

Marker Technology

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations where individuals are predominantly homozygous and loci arepredominantly dimorphic, dominant and codominant markers can be equallyvaluable. As populations become more heterozygous and multiallelic,codominant markers often become more informative of the genotype thandominant markers.

In another embodiment, markers, such as single sequence repeat markers(SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers,isozyme markers, single nucleotide polymorphisms (SNPs), insertions ordeletions (Indels), single feature polymorphisms (SFPs, for example, asdescribed in Borevitz et al. 2003 Gen. Res. 13:513-523), microarraytranscription profiles, DNA-derived sequences, and RNA-derived sequencesthat are genetically linked to or correlated with alleles of a QTL ofthe present invention can be utilized.

In one embodiment, nucleic acid-based analyses for the presence orabsence of the genetic polymorphism can be used for the selection ofseeds in a breeding population. A wide variety of genetic markers forthe analysis of genetic polymorphisms are available and known to thoseof skill in the art. The analysis may be used to select for genes, QTL,alleles, or genomic regions (haplotypes) that comprise or are linked toa genetic marker.

Herein, nucleic acid analysis methods are known in the art and include,but are not limited to, PCR-based detection methods (for example, TaqManassays), microarray methods, and nucleic acid sequencing methods. In oneembodiment, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

A method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al., 1986 Cold Spring Harbor Symp. Quant.Biol. 51:263-273; European Patent 50,424; European Patent 84,796;European Patent 258,017; European Patent 237,362; European Patent201,184; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat.No. 4,683,194), using primer pairs that are capable of hybridizing tothe proximal sequences that define a polymorphism in its double-strandedform.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; and 5,616,464, all of whichare incorporated herein by reference in their entireties. However, thecompositions and methods of this invention can be used in conjunctionwith any polymorphism typing method to type polymorphisms in corngenomic DNA samples. These corn genomic DNA samples used include but arenot limited to, corn genomic DNA isolated directly from a corn plant,cloned corn genomic DNA, or amplified corn genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening aplurality of polymorphisms. A single-feature polymorphism (SFP) is apolymorphism detected by a single probe in an oligonucleotide array,wherein a feature is a probe in the array. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464 employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of said probes to said targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group which can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is immediately adjacent to a polymorphism toincorporate a detectable nucleotide residue upon extension of theprimer. In certain embodiments, the SBE method uses three syntheticoligonucleotides. Two of the oligonucleotides serve as PCR primers andare complementary to sequence of the locus of corn genomic DNA whichflanks a region containing the polymorphism to be assayed. Followingamplification of the region of the corn genome containing thepolymorphism, the PCR product is mixed with the third oligonucleotide(called an extension primer) which is designed to hybridize to theamplified DNA immediately adjacent to the polymorphism in the presenceof DNA polymerase and two differentially labeleddideoxynucleosidetriphosphates. If the polymorphism is present on thetemplate, one of the labeled dideoxynucleosidetriphosphates can be addedto the primer in a single base chain extension. The allele present isthen inferred by determining which of the two differential labels wasadded to the extension primer. Homozygous samples will result in onlyone of the two labeled bases being incorporated and thus only one of thetwo labels will be detected. Heterozygous samples have both allelespresent, and will thus direct incorporation of both labels (intodifferent molecules of the extension primer) and thus both labels willbe detected.

In a preferred method for detecting polymorphisms, SNPs and Indels canbe detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′fluorescentreporter dye and a 3′quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

Marker-Trait Associations

For the purpose of QTL mapping, the markers included should bediagnostic of origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is very low. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander et al., (Lander et al., 1989 Genetics,121:185-199), and the interval mapping, based on maximum likelihoodmethods described therein, and implemented in the software packageMAPMAKER/QTL (Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990). Additional software includes Qgene, Version 2.23(1996), Department of Plant Breeding and Biometry, 266 Emerson Hall,XXell University, Ithaca, N.Y.). Use of Qgene software is a particularlypreferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander etal., (1989), and further described by Arús and Moreno-González, PlantBreeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak et al., 1995 Genetics, 139:1421-1428).Multiple regression methods or models can also be used, in which thetrait is regressed on a large number of markers (Jansen, Biometrics inPlant Breed, van Oijen, Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 116-124 (1994); Weber and Wricke, Advances in Plant Breeding,Blackwell, Berlin, 16 (1994)). Procedures combining interval mappingwith regression analysis, whereby the phenotype is regressed onto asingle putative QTL at a given marker interval, and at the same timeonto a number of markers that serve as ‘cofactors,’ have been reportedby Jansen et al. (Jansen et al., 1994 Genetics, 136:1447-1455) and Zeng(Zeng 1994 Genetics 136:1457-1468). Generally, the use of cofactorsreduces the bias and sampling error of the estimated QTL positions (Utzand Melchinger, Biometrics in Plant Breeding, van Oijen, Jansen (eds.)Proceedings of the Ninth Meeting of the Eucarpia Section Biometrics inPlant Breeding, The Netherlands, pp. 195-204 (1994), thereby improvingthe precision and efficiency of QTL mapping (Zeng 1994). These modelscan be extended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al., 1995 Theor. Appl.Genet. 91:33-3).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingin plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (eds.). Plenum Press, New York,pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

An F₂ population is the first generation of selfing. Usually a single F₁plant is selfed to generate a population segregating for all the genesin Mendelian (1:2:1) fashion. Maximum genetic information is obtainedfrom a completely classified F₂ population using a codominant markersystem (Mather, Measurement of Linkage in Heredity: Methuen and Co.,(1938)). In the case of dominant markers, progeny tests (e.g. F₃, BCF₂)are required to identify the heterozygotes, thus making it equivalent toa completely classified F₂ population. However, this procedure is oftenprohibitive because of the cost and time involved in progeny testing.Progeny testing of F₂ individuals is often used in map constructionwhere phenotypes do not consistently reflect genotype (e.g. diseaseresistance) or where trait expression is controlled by a QTL.Segregation data from progeny test populations (e.g. F₃ or BCF₂) can beused in map construction. Marker-assisted selection can then be appliedto cross progeny based on marker-trait map associations (F₂, F₃), wherelinkage groups have not been completely disassociated by recombinationevents (i.e., maximum disequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al., 1992 Proc. Natl. Acad.Sci. (USA) 89:1477-1481). However, as the distance between markersbecomes larger (i.e., loci become more independent), the information inRIL populations decreases dramatically.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al., 1992). Information obtained from backcross populations usingeither codominant or dominant markers is less than that obtained from F₂populations because one, rather than two, recombinant gametes aresampled per plant. Backcross populations, however, are more informative(at low marker saturation) when compared to RILs as the distance betweenlinked loci increases in RIL populations (i.e. about 0.15%recombination). Increased recombination can be beneficial for resolutionof tight linkages, but may be undesirable in the construction of mapswith low marker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al., 1991 Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular disease) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

Marker-Assisted Breeding

Further, the present invention contemplates that preferred haploidplants comprising at least one genotype of interest are identified usingthe methods disclosed in U.S. Patent Application Ser. No. 60/837,864,which is incorporated herein by reference in its entirety, wherein agenotype of interest may correspond to a QTL or haplotype and isassociated with at least one phenotype of interest. The methods includeassociation of at least one haplotype with at least one phenotype,wherein the association is represented by a numerical value and thenumerical value is used in the decision-making of a breeding program.Non-limiting examples of numerical values include haplotype effectestimates, haplotype frequencies, and breeding values. In the presentinvention, it is particularly useful to identify haploid plants ofinterest based on at least one genotype, such that only those linesundergo doubling, which saves resources. Resulting doubled haploidplants comprising at least one genotype of interest are then advanced ina breeding program for use in activities related to germplasmimprovement.

In the present invention, haplotypes are defined on the basis of one ormore polymorphic markers within a given haplotype window, with haplotypewindows being distributed throughout the crop's genome. In anotheraspect, de novo and/or historical marker-phenotype association data areleveraged to infer haplotype effect estimates for one or more phenotypesfor one or more of the haplotypes for a crop. Haplotype effect estimatesenable one skilled in the art to make breeding decisions by comparinghaplotype effect estimates for two or more haplotypes. Polymorphicmarkers, and respective map positions, of the present invention areprovided in U.S. Patent Applications 2005/0204780, 2005/0216545,2005/0218305, and Ser. No. 11/504,538, which are incorporated herein byreference in their entirety.

In yet another aspect, haplotype effect estimates are coupled withhaplotype frequency values to calculate a haplotype breeding value of aspecific haplotype relative to other haplotypes at the same haplotypewindow, or across haplotype windows, for one or more phenotypic traits.In other words, the change in population mean by fixing the haplotype isdetermined. In still another aspect, in the context of evaluating theeffect of substituting a specific region in the genome, either byintrogression or a transgenic event, haplotype breeding values are usedas a basis in comparing haplotypes for substitution effects. Further, inhybrid crops, the breeding value of haplotypes is calculated in thecontext of at least one haplotype in a tester used to produce a hybrid.Once the value of haplotypes at a given haplotype window are determinedand high density fingerprinting information is available on specificvarieties or lines, selection can be applied to these genomic regionsusing at least one marker in the at least one haplotype.

In the present invention, selection can be applied at one or more stagesof a breeding program:

a) Among genetically distinct populations, herein defined as “breedingpopulations,” as a pre-selection method to increase the selection indexand drive the frequency of favorable haplotypes among breedingpopulations, wherein pre-selection is defined as selection amongpopulations based on at least one haplotype for use as parents inbreeding crosses, and leveraging of marker-trait association identifiedin previous breeding crosses.

b) Among segregating progeny from a breeding population, to increase thefrequency of the favorable haplotypes for the purpose of line or varietydevelopment.

c) Among segregating progeny from a breeding population, to increase thefrequency of the favorable haplotypes prior to QTL mapping within thisbreeding population.

d) For hybrid crops, among parental lines from different heteroticgroups to predict the performance potential of different hybrids.

In the present invention, it is contemplated that methods of determineassociations between genotype and phenotype in haploid plants can beperformed based on haplotypes, versus markers alone (Fan et al., 2006Genetics). A haplotype is a segment of DNA in the genome of an organismthat is assumed to be identical by descent for different individualswhen the knowledge of identity by state at one or more loci is the samein the different individuals, and that the regional amount of linkagedisequilibrium in the vicinity of that segment on the physical orgenetic map is high. A haplotype can be tracked through populations andits statistical association with a given trait can be analyzed. Bysearching the target space for a QTL association across multiple QTLmapping populations that have parental lines with genomic regions thatare identical by descent, the effective population size associated withQTL mapping is increased. The increased sample size results in morerecombinant progeny which increases the precision of estimating the QTLposition.

Thus, a haplotype association study allows one to define the frequencyand the type of the ancestral carrier haplotype. An “association study”is a genetic experiment where one tests the level of departure fromrandomness between the segregation of alleles at one or more marker lociand the value of individual phenotype for one or more traits.Association studies can be done on quantitative or categorical traits,accounting or not for population structure and/or stratification. In thepresent invention, associations between haplotypes and phenotypes forthe determination of “haplotype effect estimates” can be conducted denovo, using mapping populations for the evaluation of one or morephenotypes, or using historical genotype and phenotype data.

A haplotype analysis is important in that it increases the statisticalpower of an analysis involving individual biallelic markers. In a firststage of a haplotype frequency analysis, the frequency of the possiblehaplotypes based on various combinations of the identified biallelicmarkers of the invention is determined. The haplotype frequency is thencompared for distinct populations and a reference population. Ingeneral, any method known in the art to test whether a trait and agenotype show a statistically significant correlation may be used.

Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell within the skill of the ordinary practitioner of the art.

To estimate the frequency of a haplotype, the base reference germplasmhas to be defined (collection of elite inbred lines, population ofrandom mating individuals, etc.) and a representative sample (or theentire population) has to be genotyped. For example, in one aspect,haplotype frequency is determined by simple counting if considering aset of inbred individuals. In another aspect, estimation methods thatemploy computing techniques like the Expectation/Maximization (EM)algorithm are required if individuals genotyped are heterozygous at morethan one locus in the segment and linkage phase is unknown (Excoffier etal., 1995 Mol. Biol. Evol. 12: 921-927; Li et al., 2002 Biostatistics).Preferably, a method based on the EM algorithm (Dempster et al., 1977 J.R. Stat. Soc. Ser. B 39:1-38) leading to maximum-likelihood estimates ofhaplotype frequencies under the assumption of Hardy-Weinberg proportions(random mating) is used (Excoffier et al., 1995 Mol. Biol. Evol. 12:921-927). Alternative approaches are known in the art that forassociation studies: genome-wide association studies, candidate regionassociation studies and candidate gene association studies (Li et al.,2006 BMC Bioinformatics 7:258). The polymorphic markers of the presentinvention may be incorporated in any map of genetic markers of a plantgenome in order to perform genome-wide association studies.

The present invention comprises methods to detect an association betweenat least one haplotype in a haploid crop plant and a preferred trait,including a transgene, or a multiple trait index and calculate ahaplotype effect estimate based on this association. In one aspect, thecalculated haplotype effect estimates are used to make decisions in abreeding program. In another aspect, the calculated haplotype effectestimates are used in conjunction with the frequency of the at least onehaplotype to calculate a haplotype breeding value that will be used tomake decisions in a breeding program. A multiple trait index (MTI) is anumerical entity that is calculated through the combination of singletrait values in a formula. Most often calculated as a linear combinationof traits or normalized derivations of traits, it can also be the resultof more sophisticated calculations (for example, use of ratios betweentraits). This MTI is used in genetic analysis as if it were a trait.

Any given chromosome segment can be represented in a given population bya number of haplotypes that can vary from 1 (region is fixed), to thesize of the population times the ploidy level of that species (2 in adiploid species), in a population in which every chromosome has adifferent haplotype. Identity-by-descent among haplotype carried bymultiple individuals in a non-fixed population will result in anintermediate number of haplotype and possibly a differing frequencyamong the different haplotypes. New haplotypes may arise throughrecombination at meiosis between existing haplotypes in heterozygousprogenitors. The frequency of each haplotype may be estimated by severalmeans known to one versed in the art (e.g. by direct counting, or byusing an EM algorithm). Let us assume that “k” different haplotypes,identified as “h_(i)” (i=1, . . . , k), are known, that their frequencyin the population is “f_(i)” (i=1, . . . , k), and for each of thesehaplotypes we have an effect estimate “Est_(i)” (i=1, . . . , k). If wecall the “haplotype breeding value” (BV) the effect on that populationof fixing that haplotype, then this breeding value corresponds to thechange in mean for the trait(s) of interest of that population betweenits original state of haplotype distribution at the window and a finalstate at which haplotype “h_(i)” encounters itself at a frequency of100%. The haplotype breeding value of h_(i) in this population iscalculated as:

${BV}_{i} = {{Est}_{i} - {\sum\limits_{i = 1}^{k}\; {{Est}_{i}f_{i}}}}$

One skilled in the art will recognize that haplotypes that are rare inthe population in which effects are estimated tend to be less preciselyestimated, this difference of confidence may lead to adjustment in thecalculation. For example one can ignore the effects of rare haplotypes,by calculating breeding value of better known haplotype after adjustingthe frequency of these (by dividing it by the sum of frequency of thebetter known haplotypes). One could also provide confidence intervalsfor the breeding value of each haplotypes.

The present invention anticipates that any particular haplotype breedingvalue will change according to the population for which it iscalculated, as a function of difference of haplotype frequencies. Theterm “population” will thus assume different meanings, below are twoexamples of special cases. In one aspect, a population is a singleinbred in which one intends to replace its current haplotype h_(j) by anew haplotype h_(i), in this case BV_(i)=Est_(i)-Est_(i). In anotheraspect, a “population” is a F2 population in which the two parentalhaplotype h_(i) and h_(j) are originally present in equal frequency(50%), in which case BV_(i)=½ (Est-Est).

These statistical approaches enable haplotype effect estimates to informbreeding decisions in multiple contexts. Other statistical approaches tocalculate breeding values are known to those skilled in the art and canbe used in substitution without departing from the spirit and scope ofthis invention.

In cases where conserved genetic segments, or haplotype windows, arecoincident with segments in which QTL have been identified it ispossible to deduce with high probability that QTL inferences can beextrapolated to other germplasm having an identical haplotype in thathaplotype window. This a priori information provides the basis to selectfor favorable QTLs prior to QTL mapping within a given population.

For example, plant breeding decisions could comprise:

a) Selection among haploid breeding populations to determine whichpopulations have the highest frequency of favorable haplotypes, whereinhaplotypes are designated as favorable based on coincidence withprevious QTL mapping and preferred populations undergo doubling; or

b) Selection of haploid progeny containing the favorable haplotypes inbreeding populations prior to, or in substitution for, QTL mappingwithin that population, wherein selection could be done at any stage ofbreeding and at any generation of a selection and can be followed bydoubling; or

c) Prediction of progeny performance for specific breeding crosses; or

d) Selection of haploid plants for doubling for subsequent use ingermplasm improvement activities based on the favorable haplotypes,including line development, hybrid development, selection amongtransgenic events based on the breeding value of the haplotype that thetransgene was inserted into, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

In cases where haplotype windows are coincident with segments in whichgenes have been identified it is possible to deduce with highprobability that gene inferences can be extrapolated to other germplasmhaving an identical genotype, or haplotype, in that haplotype window.This a priori information provides the basis to select for favorablegenes or gene alleles on the basis of haplotype identification within agiven population. For example, plant breeding decisions could comprise:

a) Selection among haploid breeding populations to determine whichpopulations have the highest frequency of favorable haplotypes, whereinhaplotypes are designated as favorable based on coincidence withprevious gene mapping and preferred populations undergo doubling; or

b) Selection of haploid progeny containing the favorable haplotypes inbreeding populations, wherein selection is effectively enabled at thegene level, wherein selection could be done at any stage of breeding andat any generation of a selection and can be followed by doubling; or

c) Prediction of progeny performance for specific breeding crosses; or

d) Selection of haploid plants for doubling for subsequent use ingermplasm improvement activities based on the favorable haplotypes,including line development, hybrid development, selection amongtransgenic events based on the breeding value of the haplotype that thetransgene was inserted into, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

A preferred haplotype provides a preferred property to a parent plantand to the progeny of the parent when selected by a marker means orphenotypic means. The method of the present invention provides forselection of preferred haplotypes, or haplotypes of interest, and theaccumulation of these haplotypes in a breeding population.

In the present invention, haplotypes and associations of haplotypes toone or more phenotypic traits provide the basis for making breedingdecisions and germplasm improvement activities. Non-limiting examples ofbreeding decisions include progeny selection, parent selection, andrecurrent selection for at least one haplotype. In another aspect,breeding decisions relating to development of plants for commercialrelease comprise advancing plants for testing, advancing plants forpurity, purification of sublines during development, inbred development,variety development, and hybrid development. In yet other aspects,breeding decisions and germplasm improvement activities comprisetransgenic event selection, making breeding crosses, testing andadvancing a plant through self-fertilization, using plants or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plants or parts thereof formutagenesis.

In another embodiment, this invention enables indirect selection throughselection decisions for at least one phenotype based on at least onenumerical value that is correlated, either positively or negatively,with one or more other phenotypic traits. For example, a selectiondecision for any given haplotype effectively results in selection formultiple phenotypic traits that are associated with the haplotype.

In still another embodiment, the present invention acknowledges thatpreferred haplotypes identified by the methods presented herein may beadvanced as candidate genes for inclusion in expression constructs,i.e., transgenes. Nucleic acids underlying haplotypes of interest may beexpressed in plant cells by operably linking them to a promoterfunctional in plants. In another aspect, nucleic acids underlyinghaplotypes of interest may have their expression modified bydouble-stranded RNA-mediated gene suppression, also known as RNAinterference (“RNAi”), which includes suppression mediated by smallinterfering RNAs (“siRNA”), trans-acting small interfering RNAs(“ta-siRNA”), or microRNAs (“miRNA”). Examples of RNAi methodologysuitable for use in plants are described in detail in U.S. PatentApplication Publications 2006/0200878 and 2007/0011775.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the nucleic acid molecule for a traitis transcribed into a functional mRNA molecule that is translated andexpressed as a protein product. For the practice of the presentinvention, conventional compositions and methods for preparing and usingconstructs and host cells are well known to one skilled in the art, seefor example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, ColdSpring Harbor Laboratory Press. Methods for making transformationconstructs particularly suited to plant transformation include, withoutlimitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835,4,769,061 and 4,757,011, all of which are herein incorporated byreference in their entirety. Transformation methods for the introductionof expression units into plants are known in the art and includeelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; protoplasttransformation as illustrated in U.S. Pat. No. 5,508,184; andAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301.

Another preferred embodiment of the present invention is to buildadditional value by selecting a composition of haplotypes wherein eachhaplotype has a haplotype effect estimate that is not negative withrespect to yield, or is not positive with respect to maturity, or isnull with respect to maturity, or amongst the best 50 percent withrespect to a phenotypic trait, transgene, and/or a multiple trait indexwhen compared to any other haplotype at the same chromosome segment in aset of germplasm, or amongst the best 50 percent with respect to aphenotypic trait, transgene, and/or a multiple trait index when comparedto any other haplotype across the entire genome in a set of germplasm,or the haplotype being present with a frequency of 75 percent or more ina breeding population or a set of germplasm provides evidence of itshigh value, or any combination of these.

This invention anticipates a stacking of haplotypes from multiplewindows into plants or lines by crossing parent plants or linescontaining different haplotype regions. The value of the plant or linecomprising in its genome stacked haplotype regions is estimated by acomposite breeding value, which depends on a combination of the value ofthe traits and the value of the haplotype(s) to which the traits arelinked. The present invention further anticipates that the compositebreeding value of a plant or line is improved by modifying thecomponents of one or each of the haplotypes. Additionally, the presentinvention anticipates that additional value can be built into thecomposite breeding value of a plant or line by selection of at least onerecipient haplotype with a preferred haplotype effect estimate or, inconjunction with the haplotype frequency, breeding value to which one orany of the other haplotypes are linked, or by selection of plants orlines for stacking haplotypes by breeding.

Another embodiment of this invention is a method for enhancing breedingpopulations by accumulation of one or more preferred haplotypes in a setof germplasm. Genomic regions defined as haplotype windows includegenetic information that contribute to one or more phenotypic traits ofthe plant. Variations in the genetic information at one or more loci canresult in variation of one or more phenotypic traits, wherein the valueof the phenotype can be measured. The genetic mapping of the haplotypewindows allows for a determination of linkage across haplotypes. Ahaplotype of interest has a DNA sequence that is novel in the genome ofthe progeny plant and can in itself serve as a genetic marker for thehaplotype of interest. Notably, this marker can also be used as anidentifier for a gene or QTL. For example, in the event of multipletraits or trait effects associated with the haplotype, only one markerwould be necessary for selection purposes. Additionally, the haplotypeof interest may provide a means to select for plants that have thelinked haplotype region. Selection can be performed by screening fortolerance to an applied phytotoxic chemical, such as an herbicide orantibiotic, or to pathogen resistance. Selection may be performed usingphenotypic selection means, such as, a morphological phenotype that iseasy to observe such as seed color, seed germination characteristic,seedling growth characteristic, leaf appearance, plant architecture,plant height, and flower and fruit morphology.

The present invention also provides for the screening of progeny haploidplants for haplotypes of interest and using haplotype effect estimatesas the basis for selection for use in a breeding program to enhance theaccumulation of preferred haplotypes. The method includes: a) providinga breeding population comprising at least two haploid plants wherein thegenome of the breeding population comprises a plurality of haplotypewindows and each of the plurality of haplotype windows comprises atleast one haplotype; and b) associating a haplotype effect estimate forone or more traits for two or more haplotypes from one or more of theplurality of haplotype windows, wherein the haplotype effect estimatecan then be used to calculate a breeding value that is a function of theestimated effect for any given phenotypic trait and the frequency ofeach of the at least two haplotypes; and c) ranking one or more of thehaplotypes on the basis of a value, wherein the value is a haplotypeeffect estimate, a haplotype frequency, or a breeding value and whereinthe value is the basis for determining whether a haplotype is apreferred haplotype, or haplotype of interest; and d) utilizing theranking as the basis for decision-making in a breeding program; and e)at least one progeny haploid plant is selected for doubling on the basisof the presence of the respective markers associated with the haplotypesof interest, wherein the progeny haploid plant comprises in its genomeat least a portion of the haplotype or haplotypes of interest of thefirst plant and at least one preferred haplotype of the second plant;and f) using resulting doubled haploid plants in activities related togermplasm improvement wherein the activities are selected from the groupconsisting of line and variety development, hybrid development,transgenic event selection, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

Using this method, the present invention contemplates that haplotypes ofinterest are selected from a large population of plants, and theselected haplotypes can have a synergistic breeding value in thegermplasm of a crop plant. Additionally, this invention provides forusing the selected haplotypes in the described breeding methods toaccumulate other beneficial and preferred haplotype regions and to bemaintained in a breeding population to enhance the overall germplasm ofthe crop plant.

The marker assisted breeding methods and/or methods of associatingmarkers with traits provided herein can be used with one or moreindividuals, including SSD, from any generation of plant population.Non-limiting examples of plant populations include to F1, F2, BC1,BC2F1, F3:F4, F2:F3, and so on, including subsequent filial generations,as well as experimental populations such as RILs and NILs. It is furtheranticipated that the degree of segregation within the one or more plantpopulations of the present invention can vary depending on the nature ofthe trait and germplasm under evaluation.

Plant Breeding

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). A cultivar is a race or variety of a plantspecies that has been created or selected intentionally and maintainedthrough cultivation.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) on the progeny of any cross. It isunderstood that nucleic acid markers of the present invention can beused in a MAS (breeding) program. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors such as, for example, emergence vigor, vegetativevigor, stress tolerance, disease resistance, branching, flowering, seedset, seed size, seed density, standability, and threshability etc. willgenerally dictate the choice.

Genotyping can be further economized by high throughput, non-destructiveseed sampling. In one embodiment, plants can be screened for one or moremarkers, such as genetic markers, using high throughput, non-destructiveseed sampling. In a preferred aspect, haploid seed is sampled in thismanner and only seed with at least one marker genotype of interest isadvanced for doubling. Apparatus and methods for the high-throughput,non-destructive sampling of seeds have been described which wouldovercome the obstacles of statistical samples by allowing for individualseed analysis. For example, U.S. patent application Ser. No. 11/213,430(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,431(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,432(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,434(filed Aug. 26, 2005); and U.S. patent application Ser. No. 11/213,435(filed Aug. 26, 2005), U.S. patent application Ser. No. 11/680,611(filed Mar. 2, 2007), which are incorporated herein by reference intheir entirety, disclose apparatus and systems for the automatedsampling of seeds as well as methods of sampling, testing and bulkingseeds.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredaspect, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

The development of new elite corn hybrids requires the development andselection of elite inbred lines, the crossing of these lines andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Additional data on parental lines, as well as thephenotype of the hybrid, influence the breeder's decision whether tocontinue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have most attributes of the recurrentparent (e.g., cultivar) and, in addition, the desirable traittransferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of CropImprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant Breeding Perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979; Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Manograph., 16:249, 1987; Fehr,“Principles of Variety Development,” Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376, 1987).

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.,2006 Genetics 172:663-686). This approach tracks blocks of DNA known ashaplotypes, as defined by polymorphic markers, which are assumed to beidentical by descent in the mapping population. This assumption resultsin a larger effective sample size, offering greater resolution of QTL.Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising thenucleic acid molecules of the present invention.

As used herein, a “nucleic acid molecule,” be it a naturally occurringmolecule or otherwise may be “substantially purified”, if desired,referring to a molecule separated from substantially all other moleculesnormally associated with it in its native state. More preferably asubstantially purified molecule is the predominant species present in apreparation. A substantially purified molecule may be greater than 60%free, preferably 75% free, more preferably 90% free, and most preferably95% free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “substantially purified” is not intended toencompass molecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal., 1987 Science 238:336-340; Albarella et al., European Patent144914), chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi etal., European Patent 119448).

The plant breeding methods provided herein can be used with one or moreindividuals, including SSD, from any generation of plant population.Non-limiting examples of plant populations include to F1, F2, BC1,BC2F1, F3:F4, F2:F3, and so on, including subsequent filial generations,as well as experimental populations such as RILs and NILs. It is furtheranticipated that the degree of segregation within the one or more plantpopulations of the present invention can vary depending on the nature ofthe trait and germplasm under evaluation.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1 Phenotyping for Goss' Wilt

In order to detect QTL associated with resistance to Goss' Wilt, plantswere phenotyped to determine Goss' Wilt reaction. The following ratingscale was used in order to assess resistance or susceptibility to Goss'Wilt. Phenotypic evaluations of Goss' Wilt reaction is based onpercentage of infected leaf area and rated according to a 1 (veryresistant) to 9 (susceptible) scale as provided in Table 1. Plants areartificially inoculated and visually rated approximately 3 to 4 weeksafter pollination.

TABLE 1 Disease rating scale for Goss' Wilt. Description Rating SymptomsVery Resistant 1 0% of leaf area infected; no visible lesions VeryResistant 2 ILA <1%; few lesions, dispersed through lower leavesResistant 3 1% ≦ ILA ≦ 20% Resistant 4 20% ≦ ILA ≦ 40% Mid-resistant 540% ≦ ILA ≦ 50% Mid-Susceptible 6 50% ≦ ILA ≦ 60%; lesions Susceptible 760% ≦ ILA ≦ 75% Susceptible 8 75% ≦ ILA ≦ 90% Susceptible 9 >90% offoliar area infected ILA = Infected Leaf Area

Example 2 Goss' Wilt Resistance Mapping Study 1

To examine associations between SNP markers and Goss' Wilt resistance,analyzed data from a number of studies was combined. An associationstudy was conducted to evaluate whether significant associations betweenone or marker genotypes and Goss' Wilt resistance are present in one ormore populations. In this association study, data from 10 mappingpopulations were combined. The number of individuals in the populationsranged from 186 to 369. The number of SNP markers used for screeningranged from 104 to 134. The populations were either F3 or BC1F2. A totalof 172 significant associations between SNP markers and Goss' Wiltresistance were identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, and10. The SNP markers provided can be used to monitor the introgression ofGoss' Wilt resistance into a breeding population. Significantmarker-Goss' Wilt associations are reported in FIG. 1.

Example 3 Goss' Wilt Resistance Mapping Study 2

An association study was conducted to evaluate whether significantassociations between one or marker genotypes and Goss' Wilt resistanceare present in one or more populations. In this association study, 988inbred lines were screened with 1051 SNP markers. A total of 53significant associations between SNP markers and Goss' Wilt resistancewere identified on Chromosomes 1, 2, 3, 4, 5, 6, 8, 9, and 10. The SNPmarkers provided can be used to monitor the introgression of Goss' Wiltresistance into a breeding population. SNP markers associated with Goss'Wilt resistance, level of significance, and favorable alleles arereported in FIG. 1.

Example 4 Goss' Wilt Resistance Mapping Study 3

An association study was conducted to evaluate whether significantassociations between one or more marker genotypes and Goss' Wiltresistance are present in one or more populations. In this study, arating scale of 1 to 4 was used with 1 being resistant, 2 moderatelyresistant, 3 moderately susceptible, and 4 susceptible. In thisassociation study, two F3 populations of 154 and 212 individuals werescreened with 104 SNP markers. A total of 35 significant associationsbetween SNP markers and Goss' Wilt resistance were identified onChromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The SNP markers providedcan be used to monitor the introgression of Goss' Wilt resistance into abreeding population. SNP markers associated with Goss' Wilt resistance,level of significance, and favorable alleles are reported in FIG. 1.

Example 5 Goss' Wilt Resistance Mapping Study 4

An association study was conducted to evaluate whether significantassociations between one or more marker genotypes and Goss' Wiltresistance are present in one or more populations. A population wasscreened with 518 SNP markers. A total of 80 significant associationsbetween SNP markers and Goss' Wilt resistance were identified onChromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The SNP markers providedcan be used to monitor the introgression of Goss' Wilt resistance into abreeding population. SNP markers associated with Goss' Wilt resistance,level of significance, and favorable alleles are reported in FIG. 1.

Example 6 Exemplary Marker Assays for Detecting Goss' Wilt Resistance

In one embodiment, the detection of polymorphic sites in a sample ofDNA, RNA, or cDNA may be facilitated through the use of nucleic acidamplification methods. Such methods specifically increase theconcentration of polynucleotides that span the polymorphic site, orinclude that site and sequences located either distal or proximal to it.Such amplified molecules can be readily detected by gel electrophoresis,fluorescence detection methods, or other means. Exemplary primers andprobes for amplifying and detecting genomic regions associated withGoss' Wilt resistance are given in Table 2.

TABLE 2 Exemplary assays for detecting Goss' Wilt resistance loci. SEQID SEQ ID Marker SNP Forward Reverse SEQ ID SEQ ID Marker SEQ IDPosition Primer Primer Probe 1 Probe 2 NC0027347 896 128 1332 1333 13341335 NC0071001 951 359 1336 1337 1338 1339 NC0017678 733 171 1340 13411342 1343 NC0028095 1098 116 1344 1345 1346 1347

Example 7 Oligonucleotide Hybridization Probes Useful for Detecting CornPlants with Goss' Wilt Resistance Loci

Oligonucleotides can also be used to detect or type the polymorphismsassociated with Goss' Wilt resistance disclosed herein byhybridization-based SNP detection methods. Oligonucleotides capable ofhybridizing to isolated nucleic acid sequences which include thepolymorphism are provided. It is within the skill of the art to designassays with experimentally determined stringency to discriminate betweenthe allelic state of the polymorphisms presented herein. Exemplaryassays include Southern blots, Northern blots, microarrays, in situhybridization, and other methods of polymorphism detection based onhybridization. Exemplary oligonucleotides for use in hybridization-basedSNP detection are provided in Table 3. These oligonucleotides can bedetectably labeled with radioactive labels, fluorophores, or otherchemiluminescent means to facilitate detection of hybridization tosamples of genomic or amplified nucleic acids derived from one or morecorn plants using methods known in the art.

TABLE 3 Exemplary Oligonucleotide Hybridization Probes*. Marker SNPSEQ ID Marker SEQ ID Position Probe Probe NC0027347 896 128 GCTACTAG GAAAATGG 1348 NC0027347 896 128 GCTACTAG A AAAATGG 1349 NC0071001 951 359CAACTACC T AGCATTT 1350 NC0071001 951 359 CAACTACC A AGCATTT 1351NC0017678 733 171 AGTCAAAG A TACTGCA 1352 NC0017678 733 171 AGTCAAAG CTACTGCA 1353 NC0028095 1098 116 TGCCCACA T TTGTTAT 1354 NC0028095 1098116 TGCCCACA C TTGTTAT 1355 *SNP nucleotides in bold and underlined.

Example 8 Oligonucleotide Probes Useful for Detecting Corn Plants withGoss' Wilt Resistance Loci by Single Base Extension Methods

Oligonucleotides can also be used to detect or type the polymorphismsassociated with Goss' Wilt resistance disclosed herein by single baseextension (SBE)-based SNP detection methods. Exemplary oligonucleotidesfor use in SBE-based SNP detection are provided in Table 4. SBE methodsare based on extension of a nucleotide primer that is hybridized tosequences immediately adjacent to a polymorphism to incorporate adetectable nucleotide residue upon extension of the primer. It is alsoanticipated that the SBE method can use three syntheticoligonucleotides. Two of the oligonucleotides serve as PCR primers andare complementary to the sequence of the locus which flanks a regioncontaining the polymorphism to be assayed. Exemplary PCR primers thatcan be used to type certain polymorphisms disclosed in this inventionare provided in Table 3 in the columns labeled “Forward Primer SEQ ID”and “Reverse Primer SEQ ID”. Following amplification of the regioncontaining the polymorphism, the PCR product is hybridized with anextension primer which anneals to the amplified DNA immediately adjacentto the polymorphism. DNA polymerase and two differentially labeleddideoxynucleoside triphosphates are then provided. If the polymorphismis present on the template, one of the labeled dideoxynucleosidetriphosphates can be added to the primer in a single base chainextension. The allele present is then inferred by determining which ofthe two differential labels was added to the extension primer.Homozygous samples will result in only one of the two labeled basesbeing incorporated and thus only one of the two labels will be detected.Heterozygous samples have both alleles present, and will thus directincorporation of both labels (into different molecules of the extensionprimer) and thus both labels will be detected.

TABLE 4 Probes (extension primers) for Single BaseExtension (SBE) assays. Marker SNP SEQ ID Marker SEQ ID Position ProbeProbe NC0027347 896 128 TTTTGTACTGCTACTAG 1356 NC0071001 951 359TACGGAATGCAACTACC 1357 NC0017678 733 171 GTCATGGCGAGTCAAAG 1358NC0028095 1098 116 TGGATGCTTTGCCCACA 1359

Example 9 Haploid Mapping Study for Goss' Wilt with I208993/LH287Population

The utility of haploid plants in genetic mapping of traits of interestis further demonstrated in the following example. A mapping populationwas developed for using haploid plants to map QTL associated withresistance to Goss' Wilt. The population was from the cross of inbredcorn lines 1208993 by LH287. F1 plants were induced to produce haploidseed. From the I208993/LH287 population, 1384 haploid plants wereinoculated with the Goss' Wilt pathogen and phenotyped using a truncatedrating scale of 1, 5, or 9. Ratings are done approximately 3 to 4 weeksafter pollination. Plants rated either 1 or 9 were used in the QTLmapping. By using only the extreme values (1 or 9), environmentalvariation that is inherent with disease phenotyping was reduced and abulk segregate analysis was created from which to detect major QTL.Genotyping was done using 114 SNP markers. Composite interval mappingwas conducted to examine significant associations between Goss' Wilt andSNP markers. Table 5 provides markers useful for detecting QTLassociated with resistance to Goss' Wilt in the I208993/LH287 haploidmapping population. The chromosome (Chr.) location, chromosome position(Chr. pos), and favorable (Fay.) allele are also provided in Table 5.

It is appreciated by one skilled in the art that the methods of thepresent invention can be used with one or more individuals, includingSSD, from any generation of plant population. Non-limiting examples ofplant populations include to F1, F2, BC1, BC2F1, F3:F4, F2:F3, and soon, including subsequent filial generations, as well as experimentalpopulations such as RILs and NILs. It is further anticipated that thedegree of segregation within the one or more plant populations of thepresent invention can vary depending on the nature of the trait andgermplasm under evaluation.

TABLE 5 Markers useful for detecting QTL associated with Goss' Wiltresistance in the I208993/LH287 haploid mapping population. Goss' ChrWilt Likelyhood Additive Fav. SEQ SNP Marker Chr pos. QTL ratio LODeffect allele ID Position* NC0202383 2 19 22 100.304 21.78074 0.737618 T1229 34 NC0199732 2 37 24 113.9429 24.74239 0.779994 T 1276 138NC0048553 2 46.8 25 103.8964 22.56081 0.758496 A 234 485 NC0201646 255.4 129 96.43437 20.94046 0.746649 T 1294 416 NC0201821 2 71.4 2740.13758 8.715765 0.202738 T 1295 331 NC0019110 2 75.1 27 28.411026.169374 0.173568 C 1278 153 NC0004821 3 54.4 40 47.57959 10.331780.451741 C 371 294 NC0200643 3 70.3 122 47.48045 10.31025 0.424893 C1296 106 NC0040461 4 51.2 125 80.02493 17.37719 0.620383 A 1282 366NC0034462 4 67.8 52 76.55974 16.62474 0.574876 T 1250 301 NC0200535 4132 58 29.47242 6.399855 0.142544 T 1297 411 NC0029435 4 138 58 29.251836.351953 0.139488 G 1298 551 NC0011194 5 29.3 63 27.51088 5.973912−0.227689 C 1299 218 NC0016527 5 49 66 29.15712 6.331388 −0.219392 T1255 351 NC0202055 5 76.4 68 26.18668 5.686366 −0.252002 T 1300 505NC0147719 5 160 130 47.9265 10.40711 0.492815 G 1301 48 NC0012417 5 17574 48.68852 10.57258 0.505586 T 768 137 NC0113381 6 83.8 79 28.961266.288858 −0.21407 A 850 303 NC0022200 6 93.7 80 31.16025 6.766361−0.201408 G 1302 153 NC0010347 8 69.2 131 27.38218 5.945966 −0.144382 T1015 160 NC0199582 8 86.3 99 26.24576 5.699195 −0.169537 A 1303 201 *SNPposition: refers to the position of the SNP polymorphism in theindicated SEQ ID NO.

Example 10 Haploid Mapping Study for Goss' Wilt with I208993/LH295Population

The utility of haploid plants in genetic mapping of traits of interestis further demonstrated in the following example. A mapping populationwas developed for using haploid plants to map QTL associated withresistance to Goss' Wilt. The population was from the cross of LH295 by1208993. F1 plants were induced to produce haploid seed.

From the I208993/LH295 haploid mapping population, 980 individuals werenaturally exposed to the Goss' Wilt pathogen and phenotyped using amodified rating scale of 1, 5, or 9. Plants were rated approximately 3to 4 weeks after pollination. Plants rated either 1 or 9 were used inthe QTL mapping. By using only the extreme values (1 or 9),environmental variation that is inherent with disease phenotyping wasreduced and a bulk segregate analysis was created from which to detectmajor QTL. Genotyping was done with 980 SNP markers. Table 6 providesmarkers useful for detecting QTL associated with Goss' Wilt in theI208993/LH295 haploid mapping population.

It is appreciated by one skilled in the art that the methods of thepresent invention can be used with one or more individuals, includingSSD, from any generation of plant population. Non-limiting examples ofplant populations include F1, F2, BC1, BC2F1, F3:F4, F2:F3, and so on,including subsequent filial generations, as well as experimentalpopulations such as RILs and NILs. It is further anticipated that thedegree of segregation, as well as heterozygosity, within the one or moreplant populations of the present invention can vary depending on thenature of the trait and germplasm under evaluation.

TABLE 6 Markers useful for detecting QTL associated with Goss' Wilt inthe I208993/LH295 haploid mapping population Goss' Chr. Wilt AdditiveFavorable SEQ SNP Marker Chr. pos QTL Likelihood LOD Effect Allele IDPosition* NC0199051 1 19.3 1 28.02118 6.084721 −0.22604 G 1274 141NC0105051 1 31.4 3 28.79147 6.251987 −0.236914 C 24 426 NC0032288 1133.6 10 31.20763 6.77665 0.252864 C 1275 413 NC0070305 1 166.5 1329.73574 6.457033 0.216902 A 158 532 NC0143411 2 15.4 22 31.807366.90688 −0.372898 C 218 401 NC0199732 2 37 24 51.17309 11.11209−0.506613 T 1276 138 NC0013275 2 49.7 25 56.78186 12.33002 −0.677671 T236 430 NC0199350 2 67.8 26 57.35414 12.45429 −0.577154 G 1277 226NC0019110 2 75.1 27 51.54673 11.19323 −0.633508 C 1278 153 NC0027319 293.2 29 41.90672 9.099928 −0.572435 T 272 54 NC0104528 3 24.6 3729.36476 6.376476 −0.189689 G 1247 117 NC0019963 3 40.6 39 32.035886.956503 −0.139199 C 368 1173 NC0077220 3 43.2 39 27.90631 6.059777−0.133108 A 1279 149 NC0108727 3 77.4 122 32.5836 7.075438 −0.031362 C375 241 NC0039785 3 94.5 123 30.35128 6.590696 −0.083537 T 401 512NC0031720 3 99.7 123 46.9907 10.2039 0.199348 G 408 434 NC0200377 3116.9 43 47.01889 10.21002 0.181809 A 1280 352 NC0199741 3 125.7 4428.60384 6.211245 −0.315998 A 1281 294 NC0041040 3 145.4 45 36.856578.003303 −0.551354 A 440 497 NC0055502 4 1.8 124 36.00788 7.819012−0.390433 C 498 105 NC0040461 4 51.2 125 42.90587 9.316891 −0.469569 A1282 366 NC0199420 4 102.9 55 43.93528 9.540424 −0.452635 G 1283 356NC0036240 4 112 56 38.3635 8.330528 −0.381557 A 587 441 NC0028933 4127.6 57 29.32225 6.367245 0.144007 C 599 355 NC0147712 4 136.7 5833.6318 7.303051 0.185174 A 1284 74 NC0028579 4 155.7 60 37.460128.134361 0.109588 A 629 242 NC0029487 4 171.1 126 38.35712 8.3291430.101598 G 1285 159 NC0200359 5 11.7 63 27.52949 5.977952 −0.167336 A1286 196 NC0040571 5 88.4 69 59.435 12.90615 −0.58299 C 721 154NC0017678 5 103.8 71 69.69769 15.13466 −0.722151 A 733 171 NC0083876 5124 72 29.09207 6.317263 −0.392793 T 744 513 NC0200323 5 174.8 7427.01332 5.865868 −0.253474 A 1287 181 NC0027347 7 43.8 86 57.8735412.56708 −0.542521 A 896 128 NC0201872 7 64.4 88 58.07534 12.6109−0.54188 C 1288 208 NC0145922 7 80.5 89 26.87412 5.835642 −0.271008 G940 451 NC0071001 7 99.4 90 26.59882 5.775861 −0.262452 T 951 359NC0199879 7 112.1 92 34.51543 7.494931 −0.28773 A 1289 228 NC0200055 7122.3 127 36.14355 7.848472 −0.277751 T 1290 116 NC0110771 7 138.5 9332.98577 7.162769 −0.163457 A 976 490 NC0200495 7 155.9 95 27.698126.014571 −0.118782 G 1291 302 NC0028095 9 59.4 107 29.92602 6.4983530.142796 C 1098 116 NC0144850 9 67 108 30.50354 6.62376 0.146897 G 1292244 NC0030134 10 79.4 120 27.87616 6.05323 −0.317779 TCCACTAT 1215 94NC0200312 10 85.7 128 31.10615 6.754615 −0.355789 A 1293 89 *SNPposition: refers to the position of the SNP polymorphism in theindicated SEQ ID NO.

Example 11 Introgression of Goss' Wilt Resistance Using SNP Markers

Loci associated with resistance to Goss' Wilt can be introgressed intocorn plants by methods known to those skilled in the art of plantbreeding. A plant breeder can use SNP markers to monitor theintrogression of Goss' Wilt resistance loci and to select for linescarrying the favorable allele for one or more of said SNP markers. Inthis example, the inbred line LH287 is used as a source of Goss' Wiltresistance. SNP markers used to monitor introgression of Goss' Wiltresistance loci on Chromosome 2 include NC0202383, NC0199732, NC0048553,and NC0201646 (SEQ ID NOs: 1122, 1276, 1294, and 234). SNP used tomonitor introgression of Goss' Wilt resistance loci on Chromosome 3include NC0019963 and NC0004821 (SEQ ID NOs: 368 and 371). SNP markersused to monitor the introgression of Goss' Wilt resistance loci onChromosome 4 include NC0040461 and NC0034462 (SEQ ID NOs: 1282 and1250). SNP markers used to monitor the introgression of Goss' Wiltresistance loci on Chromosome 5 include NC0147719 and NC0012417 (SEQ IDNOs: 1301 and 768). The favorable allele is the allele associated withthe resistant donor parent.

In a further illustration, the inbred line LH295 is used as a source ofGoss' Wilt resistance. SNP markers used to monitor the introgression ofGoss' Wilt resistance loci on Chromosome 2 include NC0013275, NC0199350,and NC0019110 (SEQ ID NOs: 236, 1277, and 1278). SNP markers used tomonitor the introgression of Goss' Wilt resistance loci on Chromosome 3include NC0199741 and NC0041040 (SEQ ID NOs: 1281 and 440). SNP markersused to monitor the introgression of Goss' Wilt resistance loci onChromosome 4 include NC0040461, NC0199420, and NC0036240 (SEQ ID NOs:1282, 1283, and 587). SNP markers used to monitor the introgression ofGoss' Wilt resistance loci on Chromosome 5 include NC0040571 andNC0017678 (SEQ ID NOs: 721 and 733). SNP markers used to monitor theintrogression of Goss' Wilt resistance loci on Chromosome 7 includeNC0201872 and NC0145922 (SEQ ID NOs: 1288 and 940). SNP markers used tomonitor the introgression of Goss' Wilt resistance loci on Chromosome 10include NC0200312 (SEQ ID NO: 1293). A plant breeder can use SNP markersto monitor the introgression of Goss' Wilt resistance loci and to selectfor lines carrying the favorable allele for one or more of said SNPmarkers.

The introgression of one or more resistance loci is achieved viarepeated backcrossing to a recurrent parent accompanied by selection toretain one or more Goss' Wilt resistance loci from the donor parentusing the above-described markers. This backcross procedure isimplemented at any stage in line development and occurs in conjunctionwith breeding for superior agronomic characteristics or one or moretraits of interest, including transgenic and nontransgenic traits.

Alternatively, a forward breeding approach is employed wherein one ormore Goss' Wilt resistance loci can be monitored for successfulintrogression following a cross with a susceptible parent withsubsequent generations genotyped for one or more Goss' Wilt resistanceloci and for one or more additional traits of interest, includingtransgenic and nontransgenic traits.

Example 12 Application of Markers Associated with Goss' Wilt in a CornBreeding Program

From the studies presented in FIG. 1, it is apparent that a chromosomalregion can have multiple SNP markers associated with Goss' Wiltresistance. Following are non-limiting examples of targeting at leastone marker from at least on locus associated with Goss' Wilt resistancefor the purpose of breeding corn resistant to Goss' Wilt. Specificallythe markers of the present invention have utility for generating corninbreds and hybrids resistant to Goss Wilt. The markers of the presentinvention are useful in parent selection, progeny selection, andmarker-assisted introgression and backcrossing. Exemplary markers fromChromosome 1 are NC0004909 and NC0005098 (SEQ ID NOs: 175 and 177).Exemplary markers from Chromosome 3 are NC0146497 and NC0155987 (SEQ IDNOs: 479 and 480). Exemplary markers from Chromosome 4 are NC0077408,NC0003274, and NC0009280 (SEQ ID NOs: 582, 585, and 1251). Exemplarymarkers from Chromosome 8 are NC0010392, NC0012656, and NC0008831 (SEQID NOs: 1053, 1054, and 1056).

In view of the foregoing, it will be seen that the several advantages ofthe invention are achieved and attained.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated.

Various patent and non-patent publications are cited herein, thedisclosures of each of which are, to the extent necessary, incorporatedherein by reference in their entireties.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. The breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

We claim:
 1. A method of identifying a corn plant comprising at leastone allele associated with Goss' Wilt resistance allele in a corn plantcomprising: a) genotyping at least one corn plant with at least onenucleic acid marker selected from the group consisting of SEQ ID NOs:13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97, 99, 101, 102, 106, 110, 111,119, 121, 122, 124, 128, 130-132, 136, 138, 141, 146, 153, 158-160, 162,164, 166, 169, 172, 175, 177, 186, 200, 202, 203, 207, 208, 215, 216,218, 220, 224, 228, 231-236, 244, 248, 250, 252, 256, 257, 260, 265-267,271-274, 278, 279, 282, 287, 289, 294-296, 299, 317, 320, 332-334, 337,347, 355, 362, 363, 366-368, 370, 371, 375, 381, 382, 392, 395, 401,408, 409, 411, 412, 422, 423, 429, 430, 433, 438, 440, 447, 474, 476,479, 480, 482, 486, 490, 493, 498, 500, 525, 530, 533, 556, 566, 582,585, 587, 589, 593, 594, 599, 611, 618, 621, 623, 629, 630, 632, 637,639, 646, 649, 650, 657, 665, 669, 678, 679, 688, 690, 704, 709, 710,717, 719-721, 726, 727, 733, 734, 744, 746, 758, 760, 764, 768, 773,792, 793, 812, 821, 825, 835, 844, 846, 850, 854, 856-858, 874, 876,880, 882, 885, 893, 896, 897, 915, 926, 940, 942, 949, 951, 957, 963,964, 974, 976, 981, 983, 990, 997, 999, 1000, 1015, 1016, 1027, 1043,1049, 1053, 1054, 1056, 1075, 1081, 1087, 1088, 1098-1100, 1104, 1105,1108, 1110, 1115, 1122, 1131, 1133, 1142, 1143, 1145, 1146, 1148, 1149,1159, 1168, 1174, 1184, 1186, 1196, 1204, 1212, 1215, 1229, 1234-1302,and 1303, and b) selecting at least one corn plant comprising an alleleof at least one of said markers that is associated with resistance toGoss' Wilt.
 2. The method according to claim 1, wherein the at least onecorn plant genotyped in step (a) and/or the at least one corn plantselected in step (b) is a corn plant from a population generated by across.
 3. The method of claim 2, wherein said cross is effected bymechanical emasculation, chemical sterilization, or geneticsterilization of a pollen acceptor.
 4. The method of claim 1, whereinsaid genotyping is effected in step (a) by determining the allelic stateof at least one of said corn genomic DNA markers.
 5. The methodaccording to claim 1, wherein said selected corn plant(s) of step (b)exhibit at least partial resistance to a Goss' Wilt-inducing bacteria orat least substantial resistance to a Goss' Wilt-inducing bacteria. 6.The method according to claim 1, wherein said nucleic acid marker isselected from the group consisting of SEQ ID NOs: 27, 121, 141, 175,177, 220, 224, 234, 248, 252, 381, 440, 479, 480, 533, 582, 585, 639,721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122, 1186, 1246, 1250,and
 1251. 7. The method according to claim 6, wherein said nucleic acidmarker is selected from the group consisting of SEQ ID NOs: 234 and1250.
 8. The method of claim 2, wherein said population is generated bya cross of at least one Goss' Wilt resistant corn plant with at leastone Goss' Wilt sensitive corn plant.
 9. The method of claim 2, whereinsaid population is a segregating population.
 10. The method of claim 2,wherein said cross is a back cross of at least one Goss' Wilt resistantcorn plant with at least one Goss' Wilt sensitive corn plant tointrogress Goss' Wilt resistance into a corn germplasm.
 11. The methodof claim 2, wherein said population is a haploid breeding population.12. A method of introgressing a Goss' Wilt resistance QTL into a cornplant comprising: a) screening a population with at least one nucleicacid marker to determine if one or more corn plants from the populationcontains a Goss' Wilt resistance QTL, wherein the Goss' Wilt resistanceQTL is a QTL selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, and 131 as provided in FIG. 1; and b) selecting fromsaid population at least one corn plant comprising an allele of saidmarker associated with a Grey Leaf Spot (GLS) resistance.
 13. The methodaccording to claim 12, wherein at least one of the markers is locatedwithin 5 cM of said Goss' Wilt resistance QTL.
 14. The method accordingto claim 13, wherein at least one of the markers is located within 2 cMof said Goss' Wilt resistance QTL.
 15. The method according to claim 14,wherein at least one of the markers is located within 1 cM of said Goss'Wilt resistance QTL.
 16. The method according to claim 12, wherein atleast one of the markers exhibits a LOD score of greater than 4.0 withsaid Goss' Wilt resistance QTL.
 17. The method according to claim 16,wherein said nucleic acid marker is selected from the group consistingof SEQ ID NOs: 27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 381,440, 479, 480, 533, 582, 585, 639, 721, 727, 733, 746, 768, 773, 940,1053, 1054, 1122, 1186, 1246, 1250, and
 1251. 18. The method accordingto claim 17, wherein said nucleic acid marker is selected from the groupconsisting of SEQ ID NOs: 234 and
 1250. 19. The method of claim 12,wherein said population is a segregating population.
 20. A corn plantobtained by the method of claim 1, wherein said corn plant comprises atleast one allele of a nucleic acid marker selected from the groupconsisting of SEQ ID NOs: 13, 19, 24, 27, 36, 50, 53, 90, 94, 95, 97,99, 101, 102, 106, 110, 111, 119, 121, 122, 124, 128, 130-132, 136, 138,141, 146, 153, 158-160, 162, 164, 166, 169, 172, 175, 177, 186, 200,202, 203, 207, 208, 215, 216, 218, 220, 224, 228, 231-236, 244, 248,250, 252, 256, 257, 260, 265-267, 271-274, 278, 279, 282, 287, 289,294-296, 299, 317, 320, 332-334, 337, 347, 355, 362, 363, 366-368, 370,371, 375, 381, 382, 392, 395, 401, 408, 409, 411, 412, 422, 423, 429,430, 433, 438, 440, 447, 474, 476, 479, 480, 482, 486, 490, 493, 498,500, 525, 530, 533, 556, 566, 582, 585, 587, 589, 593, 594, 599, 611,618, 621, 623, 629, 630, 632, 637, 639, 646, 649, 650, 657, 665, 669,678, 679, 688, 690, 704, 709, 710, 717, 719-721, 726, 727, 733, 734,744, 746, 758, 760, 764, 768, 773, 792, 793, 812, 821, 825, 835, 844,846, 850, 854, 856-858, 874, 876, 880, 882, 885, 893, 896, 897, 915,926, 940, 942, 949, 951, 957, 963, 964, 974, 976, 981, 983, 990, 997,999, 1000, 1015, 1016, 1027, 1043, 1049, 1053, 1054, 1056, 1075, 1081,1087, 1088, 1098-1100, 1104, 1105, 1108, 1110, 1115, 1122, 1131, 1133,1142, 1143, 1145, 1146, 1148, 1149, 1159, 1168, 1174, 1184, 1186, 1196,1204, 1212, 1215, 1229, 1234-1302, and 1303, wherein said allele isassociated with Goss' Wilt resistance.
 21. The corn plant according toclaim 20, wherein said nucleic acid marker is selected from the groupconsisting of SEQ ID NOs: 27, 121, 141, 175, 177, 220, 224, 234, 248,252, 381, 440, 479, 480, 533, 582, 585, 639, 721, 727, 733, 746, 768,773, 940, 1053, 1054, 1122, 1186, 1246, 1250, and
 1251. 22. The cornplant of claim 20, wherein the corn plant exhibits at least partialresistance to a Goss' Wilt-inducing bacterium or at least substantialresistance to a Goss' Wilt-inducing bacterium.
 23. The corn plantaccording to claim 20 a nucleic acid marker selected from the groupconsisting of SEQ ID NOs: 234 and
 1250. 24. The corn plant of claim 20,wherein said corn plant is a haploid corn plant.
 25. A corn plantobtained by the method of claim 12, wherein said corn plant comprises aGoss' Wilt resistance QTL selected from the group consisting of QTLnumbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, and 131 as provided inFIG.
 1. 26. An isolated nucleic acid molecule for detecting a molecularmarker representing a polymorphism in corn DNA, wherein said nucleicacid molecule comprises at least 15 nucleotides that include or areimmediately adjacent to said polymorphism, wherein said nucleic acidmolecule is at least 90 percent identical to a sequence of the samenumber of consecutive nucleotides in either strand of DNA that includeor are immediately adjacent to said polymorphism, and wherein saidmolecular marker is selected from the group consisting of SEQ ID NOs:27, 121, 141, 175, 177, 220, 224, 234, 248, 252, 440, 479, 480, 533,582, 585, 639, 721, 727, 733, 746, 768, 773, 940, 1053, 1054, 1122,1186, 1234-1302, and
 1303. 27. The isolated nucleic acid according toclaim 26, wherein said molecular marker is selected from the groupconsisting of SEQ ID NOs: 27, 121, 141, 175, 177, 220, 224, 234, 248,252, 381, 440, 479, 480, 533, 582, 585, 639, 721, 727, 733, 746, 768,773, 940, 1053, 1054, 1122, 1186, 1246, 1250, and
 1251. 28. The isolatednucleic acid of claim 27, wherein said molecular marker is selected fromthe group consisting of SEQ ID NOs: 1250.